System, method and devices for tissue-based diagnosis

ABSTRACT

Disclosed are kits for at least partly liquefying tissue. The kit may include a liquefaction promoting medium (LPM). The LPM may include a non-ionic surfactant; a zwitterionic surfactant; and an abrasive material. The kit may include instructions. The instructions may direct a user to treat a tissue of a living subject by: applying the LPM together with the abrasive material to the tissue of the living subject; and transmitting energy to the tissue of the living subject through the abrasive material in the presence of the LPM effective to cause at least partial dissolution of one or more components of the tissue of the living subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 14/591,792, filed Jan. 7, 2015, which is acontinuation application of U.S. patent application Ser. No. 13/126,105,filed Apr. 26, 2011, which is a National Stage Entry of InternationalApplication Ser. No. PCT/US2010/024010, filed Feb. 12, 2010; whichclaims priority from U.S. Provisional Patent Application Ser. No.61/152,585, filed Feb. 13, 2009, each of which is entirely incorporatedby reference herein.

Reference to the Sequence Listing

The Sequence Listing submitted as a text file named“UCSB_2009_490_9_(DXB_77USCON)_SequenceListing_ST25” created on Aug. 17,2015, and having a size of 1,658 bytes is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

The biomolecular composition of human tissues, represented by amultitude of lipids, proteins, nucleic acids, and other miscellaneousmolecules, is a sensitive indicator of local pathologies, such ascancer, allergies, and eczema, as well as several systemic diseases,such as cardiovascular disease, Alzheimer's disease, and diabetes. Inaddition, tissue molecular composition also holds critical informationabout the body's exposure to exogenous chemical and biological entities.However, this information is not currently used in diagnostic methodsdue to a lack of patient-friendly and standardized methods for routinesample collection from tissues. Instead, clinical diagnosis isinvariably performed by visual observation and histopathologicalanalysis of tissue biopsies, which are highly limited due to theirqualitative nature, leading to increased misdiagnosis and inappropriateuse. In addition to being invasive, current methods also fall short inexplaining a complete molecular genesis of diseases, and fail todistinguish between diseases.

Prior approaches using physical and chemical methods for assessingtissue fluid have focused chiefly on extracting a few low molecularweight molecules that are freely present in the interstitial fluid, suchas calcium and glucose. Use of tape stripping for physically harvestingsuperficially-lying tissue constituents with an adhesive tape has beenreported; however this technique has been shown to be limited byinefficacy, lack of a standardized protocol, and high heterogeneity intissue sampling.

BRIEF SUMMARY OF THE INVENTION

The current invention describes system, method and device, as well ascompositions useful in such systems, methods and devices, involvingapplication of energy to a tissue of interest to generate a liquefiedsample comprising tissue constituents so as to provide for rapid tissuesampling, as well as qualitative and/or quantitative detection ofanalytes that may be part of tissue constituents (e.g., several types ofbiomolecules, drugs, and microbes) Determination of tissue compositioncan be used in a variety of applications, including diagnosis orprognosis of diseases, evaluating bioavailability of therapeutics indifferent tissues following drug administration, forensic detection ofdrugs-of-abuse, evaluating changes in the tissue microenvironmentfollowing exposure to a harmful agent, tissue decontamination andvarious other applications.

The current invention provides methods and devices for generating aliquefied tissue sample from a subject—living or deceased. The deviceand method involve applying energy and a liquefaction promoting mediumto a tissue of interest of a subject, the applying producing a liquefiedtissue sample, and collecting the liquefied tissue sample. In someembodiments, an analysis for the presence or absence of at least oneanalyte in the liquefied tissue sample is performed, wherein theanalysis facilitates diagnosis of a condition of interest. In certainembodiments, the analysis involves generating an analyte profile fromthe liquefied tissue sample and comparing the analyte profile to areference analyte profile, wherein the comparing facilitates diagnosisof a condition of interest.

In some embodiments, the purpose of said tissue liquefaction is toremove, or decontaminate the tissue from undesired substances.Non-limiting examples of such undesired substances include chemicals,environmental contaminants, biological toxins, and in general substancesthat are considered toxic or hazardous to the body. In certainembodiments, the said method of decontamination is performed bycontinuously moving the tissue liquefaction device overtissue-of-interest until removal of undesired substances at a preferredlevel is attained.

In some embodiments, the liquefaction promoting agent comprises of oneof more of sodium chloride, potassium chloride, sodium phosphatedibasic, potassium phosphate monobasic,3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid,N,N-bis(2-hydroxyethyl)glycine, tris (hydroxymethyl) methylamine,N-tris(hydroxymethyl)methylamine, N-tris(hydroxymethyl)methylglycine,4,2-hydroxyethyl-1-piperazineethanesulfonic acid,2-{[tris(hydroxymethyl)methyl)methyl]amino}ethanesulfonic acid,3-(N-morpholino)propanesulfonic acid,piperazine-N,N′-bis(2-ethanesulfonic acid), dimethylarsinic acid, salinesodium citrate, 2-(N-morpholino)ethanesulfonic acid. In certainembodiments, the liquefaction promoting agent comprises of one or moreof a protease inhibitor, an RNase inhibitor, or a DNase inhibitor. Incertain embodiments, the liquefaction promoting agent comprises at leastone of free radical scavenger, a defoaming agent, and a proteinstabilizer. In certain embodiments, the liquefaction promoting agentcomprises at least one of Brij-30, 3-(decyl dimethyl ammonio) propanesulfonate (DPS), 3-(dodecyl dimethyl ammonio) propane sulfonate (DDPS),N-lauroyl sarcosine (NLS), Triton X-100, Sodium dodecyl sulfate, DMSO,fatty acids, azone, EDTA, or sodium hydroxide. In certain embodiments,the liquefaction promoting agent comprises a suspension of abrasiveparticles. In certain embodiments, the abrasive particles comprisesilica or aluminum oxide.

In some embodiments, the energy is applied in the form of ultrasound,mechanical, optical, thermal, or electrical energy. In certainembodiments, the mechanical energy is applied by an abrasive material.In certain embodiments, the thermal energy is applied in the form ofradio frequency energy. In certain embodiments, the optical energy isapplied in the form of a laser.

In some embodiments, the liquefied tissue sample is generated for eachof a healthy tissue of interest of the subject and a suspected diseasedtissue of interest of the subject, and the analysis comprises comparinganalytical results from the healthy tissue sample with analyticalresults from the suspected diseased tissue sample, wherein the comparingfacilitates diagnosis of a condition of interest. In some embodiments,the liquefied tissue sample is generated for multiple tissue sites andthe analysis comprises comparing analytical results from the multipletissue sites, wherein said comparing facilitates diagnosis of acondition of interest. In some embodiments, the liquefied tissue sampleis collected from multiple tissue sites, and the samples are combined tomake a diagnosis.

In some embodiments, the liquefied tissue sample is collected byaspiration. In certain embodiments, the collecting is by retaining theliquefaction agent in a housing placed in contact with the tissue. Incertain embodiments, the collecting is by mechanized transfer of theliquefied tissue sample in a housing located in the device.

In some embodiments, the liquefied tissue sample is mixed with asubstance which assists in further liquefaction and in stabilization ofanalytes of interest for storage or transportation. In certainembodiments, the transferred tissue sample from that sample container ismixed with the substances which are pre-stored in a container. Examplesinclude a protein stabilizer such as protease inhibitor, a nucleic-acidstabilizer such as EDTA phenol, nonspecific proteinase, an RNaseinhibitor and a DNase inhibitor, a defoaming agent, and surfactants suchas Triton X-100, sodium dodecyl sulfate, and DMSO, and abrasiveparticles comprise silica or aluminum oxide.

In certain embodiments, the device evaluates the tissue of interestprior, during, or after liquefaction process. In certain embodiments,the evaluation is performed by electrochemical, biochemical, or opticalmeans. In some embodiments, the evaluation involves measurement oftissue's electrical conductivity. In an exemplary embodiment, electricalconductivity is measured by a means applying an AC electrical signalacross the tissue of interest. The said electrical signal has voltagebetween 0.1 mV and 10 V and frequency between 1 Hz and 100 kHz.

In some embodiments, the device involves detecting certain tissueconstituents in the liquefied tissue sample prior to analysis of ananalyte of interest, such as a disease marker. In certain embodiments,the detecting is by electrochemical, biochemical, or optical means. Insome embodiments the electrochemical means of detecting is anion-elective electrode. In some embodiments the optical means ofdetecting is measuring the absorption or scattering coefficient of aliquid solution.

In some embodiments, the energy is applied to a tissue in the form ofultrasound with a mechanical index between 0.1 and 50. In certainembodiments, the energy is applied by contacting the tissue with amoving abrasive surface. In certain embodiments, the energy is appliedto the tissue by contacting the tissue with a moving brushing devicecomprising a plurality of bristles. In certain embodiments, the energyis applied to the tissue by mechanical insertion of a patch bearingplurality of micro-needles into the tissue; and further injection ofliquefaction medium through the micro-needles into the tissue. In someembodiments, additional energy is applied by moving the saidmicro-needle patch after its insertion into the tissue. In certainembodiments, the energy is applied to the tissue by mechanized stirringof the liquefaction agent. In certain embodiments, the energy is appliedto the tissue by contacting the tissue with a high velocity jetcomprising of liquefaction promoting medium, which may also containabrasive particles in different embodiments.

In some embodiments, the tissue comprises breast, prostate, eye, vagina,bladder, nail, hair, colon, testicles, or intestine. In certainembodiments, the tissue comprises skin or a mucosal membrane. In certainembodiments, the tissue comprises lung, brain, pancreas, liver, heart,bone, or aorta wall.

In sonic embodiments, the analyte comprises a small molecule, a drug ormetabolite thereof, a polypeptide, a lipid, a nucleic acid, or amicrobe. In certain embodiments, the analyte comprises an antibody, acytokine, an illicit drug, or a cancer biomarker.

In some embodiments, the liquefied tissue sample is held in a container,and the analyte profile is generated by integrating the liquid containerwith one or more analytical devices. In certain embodiments, the tissueliquefaction device contains a means for measuring the concentration ofa calibrator analyte to provide a means for calibrating the analysis ofthe analyte.

In some embodiments, the device involves diagnosing allergic disease ina subject, and the device comprises means for analyzing the liquefiedtissue sample for the presence or absence of IgE and IgG antibodies,cytokines such as IL4, IL5, IL-10, IL-12, IL13, IL-16, GM-CSF, RANTES,MCP-4, CTACK/CCL27, IFN-g, TNFa, CD23, CD-40, Eotaxin-2, and TARC,wherein the analysis facilitates diagnosis of allergic disease in thesubject.

In some embodiments, the device involves diagnosing cancer in a subject,and the device comprises means for analyzing the liquefied tissue samplefor the presence or absence of one or more cancer markers, wherein theanalysis facilitates diagnosis of cancer in the subject. In certainembodiments, the tissue of interest is breast, colon, prostate, skin,testicle, intestine, or mouth.

In some embodiments, the device involves diagnosing heart disease in asubject, and the device comprises means for analyzing the liquefiedtissue sample for the presence or absence of one or more of cholesterol,triglycerides, lipoproteins, free fatty acids, and ceramides, whereinthe analysis facilitates diagnosis of heart disease in the subject.

In some embodiments, the device involves detecting the presence of anillicit drug, or metabolite thereof, in a subject, and the devicecomprises means for analyzing the liquefied tissue sample for thepresence or absence of an illicit drug, or metabolites thereof, whereinthe analysis provides for detection of illicit drugs in the subject.

In some embodiments, the device involves detecting a microorganism in asubject, and the device comprises means for applying energy and aliquefaction medium to a tissue of interest in a subject and analyzingthe liquefaction medium for the presence or absence of a microorganism,wherein the analysis provides for detection of the presence or absenceof a microorganism.

Another object of the current invention is to provide a method anddevice for liquefying a tissue of a subject for facilitating the passageof a drug across or into the tissue. The method and device disclosedabove are applicable not only to collection of tissue constituents butalso to drug delivery. The device and method involve applying energy anda liquefaction medium to a tissue of interest of a subject, anddelivering a drug through or into the site of the tissue to beliquefied. The advantage of using the present invention is 1) to providehigher fluxes of drugs into a tissue, and 2) to allow greater control offluxes into a tissue. Drugs which would simply not pass through thetissues such as the skin are forced through the tissues when the methodis applied.

In some embodiments, the present invention offers a method fordelivering one or more drugs through the tissue to be liquefied into thecirculatory system, which circumvents degradation in thegastrointestinal tract and rapid metabolism by the liver from whichdrugs to be routinely administered either orally or by injection suffer.In certain embodiments, the current invention provides a method anddevice for delivering one or more drugs locally to the tissue ofinterest, thus limiting side effects to the healthy tissues. The methodand device may also be applicable for enhancing transport to cellularmembranes.

In particular, the device of the present invention consists of thefollowing major components: 1) an energy generator; 2) a liquefactionpromoting medium; 3) a reservoir to hold drugs to be delivered and/ orcollect the liquefied tissue sample.

A drug to be administered can be added into the liquefaction mediumprior or during tissue liquefaction process. In an alternate embodiment,application of energy is in combination of the liquefaction medium whichdoes not contain a drug can be used for liquefying a tissue, andsubsequently a drug in an appropriate carrier such as a patch can beapplied on a site of the tissue to be liquefied.

The transport of drug into the tissue can be further enhanced by thesimultaneous or subsequent application of a secondary driving force suchas chemical permeability or transport enhancers, convection, osmoticpressure gradient, concentration gradient, iontophoresis,electroporation, magnetic field, ultrasound, or mechanical pressure. Thedriving force can be applied continuously over a period of time or atintervals during the period of liquefaction.

In some embodiments, the tissue to be administered comprises an organsas well as biological surfaces in certain embodiment, the biologicalsurfaces comprise a biological membrane and cellular membrane. Incertain embodiment, the biological membrane comprises skin or a mucosalmembrane. In certain embodiments, the biological membrane comprises abuccal membrane, eye, vagina, colon, or intestine. In some embodiment,the tissue comprises a diseased tissue.

In one embodiment, a device is provided that can be used on a tissue toobtain a liquefied sample comprising an energy source operably coupledto the tissue, and a chamber, operably coupled to said tissue, capableof delivering liquefaction promoting medium to and/or collecting saidliquefied sample from said tissue.

In another embodiment, the device can be used on a tissue which is apart of a living organism; and the tissue can be excised from theorganism prior to diagnosis.

In another embodiment, the device of claim 1 wherein the liquefiedtissue sample is transferred to an assay for monitoring the presence orabsence of at least one analyte.

In yet another embodiment, the chamber of the device can be asponge-bellow assembly where the sponge is capable of storing saidliquefaction promoting medium and/or liquefied tissue sample.

In another embodiment, a device is provided comprising an energy sourceoperably coupled to the tissue, and a chamber, operably coupled to saidtissue, capable of delivering liquefaction promoting medium to and/orcollecting said liquefied sample from said tissue; also comprises atube/needle, connected to said chamber, capable of delivering theliquefaction promoting medium to and/or aspirating liquefied tissuesample from the tissue.

In still another embodiment, a device is provided comprising an energysource operably coupled to the tissue, and a chamber, operably coupledto said tissue, capable of delivering liquefaction promoting medium toand/or collecting said liquefied sample from said tissue; also comprisesa sample container, operably connected to said chamber, capable ofstoring aspirated liquefied tissue sample containing analytes, ortransferring said aspirated liquefied tissue sample to an ancillarychamber; wherein the chamber is used only to deliver the liquefactionpromoting medium to the chamber.

In another embodiment, a pressurized container and/or vacuum containeris part of the device, which facilitates transfer of said liquefactionpromoting medium and/ or liquefied tissue sample.

In one embodiment, the energy emitted from the energy source in thedevice is in the form of ultrasound, mechanical, optical, thermal, orelectrical energy. In a particular embodiment, the mechanical energy isapplied to the tissue by an abrasive material, vacuum, pressure or shearforce. In another embodiment, the thermal energy is applied to thetissue in the form of radio frequency energy. In another embodiment, theoptical energy is applied to the tissue in the form of a laser.

In yet another embodiment, a device is provided comprising an energysource operably coupled to the tissue, and a chamber, operably coupledto said tissue, capable of delivering liquefaction promoting medium toand/or collecting said liquefied sample from said tissue furthercomprising a sample container, operably connected to said chamber,capable of storing aspirated liquefied tissue sample containinganalytes, or transferring said aspirated liquefied tissue sample to anancillary chamber; wherein the chamber is used only to deliver theliquefaction promoting medium to the chamber.

In another embodiment, a device is provided comprising an energy sourceoperably coupled to the tissue, and a chamber, operably coupled to saidtissue, capable of delivering liquefaction promoting medium to and/orcollecting said liquefied sample from said tissue, wherein the energysource comprises of a pad connected to a shaft.

In a more particular embodiment, the shaft has a pressure sensing unit,which maintains a predetermined pressure profile on to the tissue uponcontact.

In another embodiment, the pad is selected from a group consisting of anabrasive surface and a patch comprising of a plurality of micro-needles.

In yet another embodiment, the device further comprises a plunger,operably connected to the top of the chamber.

In another embodiment, the device is divided into an upper and lowerunit, and wherein the lower unit is detachable from said upper unit;wherein the upper unit comprises the energy source and the lower unitcomprises the chamber.

In still another embodiment, the device further comprises an analyticalunit operably connected to the chamber, and where the analytic unit iscapable of performing temporal monitoring of the tissue sample byelectrochemical, biochemical or optical means; or the analytic unit iscapable of analyzing the analytes within said liquefied tissue sample.

In another embodiment, the device is connected to a diagnostic probe ora catheter; wherein the diagnostic probe is selected from a groupconsisting of endoscope, colonoscope, and laparoscope.

In still another embodiment, the use of the device results in situliquefaction of the tissue sample.

In another embodiment, the device contains a liquefaction promotingmedium that can preserve and enhance the detection of proteins, lipidsand nucleic acids, comprising: 3-(decyl dimethyl ammonio) propanesulfonate (DPS) and polyethylene glycol dodecyl ether (Brij 30)dissolved in a buffered solution; and where the concentration of3-(decyl dimethyl ammonio) propane sulfonate and polyethylene glycoldodecyl ether (B30) is between 0.01-10% (w/v); and where the 3-(decylmethyl ammnonio) propane sulfonate and polyethylene glycol dodecyl etherare present at a ratio of 50:50.

In yet another embodiment, the liquefaction promoting medium within thedevice is buffered in a solution comprising either phosphate-bufferedsaline, tris-buffered saline, tris-HCl or EDTA.

In another embodiment, liquefaction promoting medium within the devicecomprises a nonionic surfactant selected from a Brij series surfactant,a Triton-X surfactant, and a Sorbitan surfactant; an anionic or azwitterionic surfactant; and a hydrophilic solvent; wherein the mediumhas a total concentration of the surfactants from about 0.01%-10% (w/v).

These and other features of the invention will become apparent to thosepersons skilled in the art upon reading the details of the system,method and device for tissue-based diagnosis as more fully describedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.included in the drawings are the following figures:

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G are cross-sectional drawingsillustrating structure, components, and function of various abrasiveenergy-based tissue liquefaction devices.

FIGS. 1A, 1B, 1C, 1C and 1E, 1F, and 1G show the sequential working oftwo separate liquefaction devices.

FIG. 1A demonstrates a tissue liquefaction device prior to operation.

FIG. 1B demonstrates a tissue liquefaction device in operation while incontact with a tissue.

FIG. 1C demonstrates a tissue liquefaction device post operation.

FIG, 1D is a schematic representation of a pressure-sensitive motorizedshaft bearing an abrasive head.

FIG. 1E demonstrates a tissue liquefaction device prior to operation.

FIG. 1F demonstrates a tissue liquefaction device in operation while incontact with a tissue.

FIG. 1G demonstrates a tissue liquefaction device post operation.

FIGS. 2A and 2B are cross-sectional drawings of moveable tissueliquefaction devices for continuous sampling of a large area of tissues.

FIG. 2A shows a device that utilizes a rotary abrasive component asmeans for applying mechanical energy to tissues for liquefaction.

FIG. 2B shows a device that utilizes a piezoelectric element as meansfor applying mechanical energy to tissues for liquefaction.

FIGS. 3A, 3, 3C, 3D, and 3E are cross-sectional drawings illustratingstructure and components of various linear abrasive motion-based tissueliquefaction devices.

FIG. 3A shows a front view of an example liquefaction device including arack and pinion arrangement.

FIG. 3B shows a side view of an example liquefaction device including arack and pinion arrangement.

FIG. 3C shows a front view of an example liquefaction device includingmultiple rack and pinion arrangements.

FIG. 3D shows a side view of an example liquefaction device includingmultiple rack and pinion arrangements.

FIG. 3E is a schematic representation of a pressure-sensitive supportshaft bearing a gear.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, and 4G are cross-sectional drawings illustseveral types of abrasive heads.

FIG. 4A illustrates an abrasive component including a sheet of abrasivematerial with uniform thickness.

FIG. 4B illustrates an abrasive component a disk of abrasive materialwith gradient abrasiveness.

FIG. 4C illustrates an abrasive component with a smooth and roundedtissue-facing surface.

FIG. 4D illustrates a circular ring-shaped abrasive component.

FIG. 4E illustrates an abrasive component including a brush withbristles of uniform height and abrasiveness.

FIG. 4F illustrates a circular disc-shaped brush with bristles of highabrasiveness at the center surrounded by bristles with low abrasivenessin the disc periphery.

FIG. 4G illustrates a brush with bristles of different lengths forming asmooth and rounded tissue-facing surface.

FIGS. 5A, 5B, 5C, and 5D are cross-sectional device drawings andschematics for measuring electrical conductivity of tissues.

FIG. 5A illustrates a measurement electrode located as an inner surfacelining of a UM housing.

FIG. 5B illustrates a reference electrode as an extension of a LPMhousing placed in peripheral vicinity of a region of the tissue beingliquefied.

FIG. 5C illustrates a reference handheld cylindrical electrodeelectrically connected with electrical conductivity measurementcomponents located in a liquefaction device.

FIG. 5D illustrates a reference patch electrode electrically connectedwith electrical conductivity measurement components located in aliquefaction device.

FIGS. 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, 6I, and 6J are a collection ofcross-sectional drawings illustrating structure, components andfunctioning of various microneedle-based tissue liquefaction devices.

FIG. 6A illustrates a microneedle patch bearing a multitude ofmicroneedles pre-filled with LPM.

FIG. 6B illustrates a vibratory component secured on a microneedlepatch.

FIG. 6C illustrates the elements of a vibratory component.

FIG. 6D illustrates a flexible elastic cap fitted on top of a patch toreplace a housing; the flexible elastic cap being pushed in to injectLPM.

FIG. 6E illustrates a flexible elastic cap fitted on top of a patch toreplace a housing; the flexible elastic cap being pushed out to withdrawLPM.

FIG. 6F illustrates microneedles coated with a substance to enhancetissue liquefaction.

FIG. 6G illustrates an electromagnet placed on top of patch to effectlinear oscillatory motion of microneedles; oscillatory motion beingoutward.

FIG. 6H illustrates an electromagnet placed on top of patch to effectlinear oscillatory motion of microneedles; oscillatory motion beinginward.

FIG. 6I illustrates electromagnets placed symmetrically around patch toeffect rotary motion of microneedles.

FIG. 6J illustrates electromagnets placed symmetrically around patch toeffect rotary motion of microneedles; rotatory motion at a 90° turn fromthe orientation depicted in FIG. 6I.

FIGS. 7A, 7B, 7C, 7D, 7E, and 7F are cross-sectional drawings of anexemplary abrasive energy-based tissue liquefaction device.

FIG. 7A illustrates an example abrasive energy-based tissue liquefactiondevice including various assembly components of the device.

FIGS. 7B, 7C, and 7D show sequential working steps of the device.

FIGS. 7B and 7C show transfer of the liquefaction medium to be incontact with tissue.

FIG. 7B illustrates the assembled device placed against a portion ofskin such that an abrasive pad faces the portion of skin.

FIG. 7C illustrates sample generation by liquefaction.

FIG. 7D illustrates collection of the sample in a container.

FIGS. 7E and 7F show post- liquefaction retrieval of sampling containerfrom the device.

FIG. 7E illustrates a device component including a sample containerremoved from the device.

FIG. 7F illustrates removal of a sample container from a devicecomponent.

FIGS. 8A, 8B, 8C, and 8D are cross-sectional drawings illustratingsequential working steps of an exemplary microneedle-based tissueliquefaction device.

FIGS. 8A and 8B illustrate transfer of the liquefaction medium to beplaced in contact with the tissue.

FIG. 8A illustrates a device placed against a portion of skin such thata microneedle bearing patch is facing the portion of skin.

FIG. 8B illustrates a sliding plunger pushed towards a skin tissue suchthat a I-PM soaked sponge is squeezed and releases LPM into a housing.

FIG. 8C illustrates sample generation by liquefaction: a sliding plungeris further pushed into a skin tissue leading to insertion ofmicroneedles into the skin tissue.

FIG. 8D illustrates collection of the sample in a container: apre-vacuumized sample container is pushed towards a skin tissue suchthat a needle punctures the sample container resulting in aspiration ofa sample from housing.

FIGS. 9A, 9B, 9C, and 9D are drawings illustrating a sampling containerand show the sequential working steps for transporting and/or analysisof the generated samples.

FIG. 9A illustrates substrates coated on the inside surface of acontainer, the substrates effective to selectively bind analytes ofinterest.

FIG. 9B illustrates analytes in a liquefied tissue sample selectivelycaptured by substrates coated on the inside surface of a container.

FIG. 9C illustrates sample material discarded from a container whileanalytes remain in the container, the analytes being bound to substratescoated on the inside surface of a container.

FIG. 9D illustrates analytes eluted by a buffer from substrates coatedon the inside surface of a container for subsequent analysis.

FIGS. 10A, 10B, and 10C are graphs illustrating the screening odologyfor identifying unique surfactant formulations of LPMs.

FIG. 10A shows a graph ranking over 150 surfactant formulations in theirability to preserve protein bioactivity.

FIG. 10B shows a graph ranking best formulations from FIG. 10A on theirtissue solubilization potential.

FIG. 10C shows a graph comparing the best LPM from entire screening 0.5%(w/v) DPS-Brij30 with other conventional surfactants in their potentialto sample functional proteins from skin tissue.

FIGS. 11A and 11B are graphs illustrating LPM-assisted preservation ofbioactivities of various proteins.

FIG. 11A shows a graph illustrating the bioactivity of IgE.

FIG. 11B shows a graph illustrating the fractional bioactivites of IgE,LDH and β-gal under mechanical stress of ultrasound exposure.

FIGS. 12A, 12B, and 12C are drawings illustrating the effect ofultrasonic exposure in the presence of LPM (saline solution 010.5% (w/v)DPS-B30) to sample a variety of functional disease biomarkers.

FIG. 12A shows a graph illustrating the effect of ultrasonic exposure inthe presence of LPM for allergen-specific IgE antibodies.

FIG. 12B shows a graph illustrating the effect of ultrasonic exposure inthe presence of LPM for cholesterol.

FIG. 12C shows a graph illustrating the effect of ultrasonic exposure inthe presence of LPM for bacteria.

FIG. 13 is a graph illustrating the effect of buffers in LPMs on thecompatibility with quantitative PCR.

FIG. 14 is a graph illustrating the influence of surfactant mixtures onthe compatibility with quantitative PCR.

FIG. 15 is a graph illustrating the effect of ultrasound intensity andexposure time on E. Coli viability. Samples were exposed to ultrasoundat intensities of 1.7 W/cm² () and 2.4 W/cm² (▪). Each point representsthe mean value from three independent samples.

FIG. 16 is a photograph of agarose gel-electrophoresis of genornic DNAfrom E. coli cells sonicated at different conditions in tris-HCl. Lane1=molecular standard; lane 2=non-treated cell; lane 3=1.7 W/cm², 2 min;lane 4=1.7 W/cm² 3 min; lane 5=2.4 W/cm², 3 min.

FIGS. 17A and 17B are graphs illustrating the number of bacteria sampledby ultrasound coupling with tris-HCl, swabbing, and surfactant scrubtechnique. Each point represents the mean value from five independentsamples.

FIG. 17A is a graph illustrating the number of bacteria sampled(CFU/cm²) by ultrasound coupling with tris-HCl, swabbing, and surfactantscrub technique—measured by culture assay.

FIG. 17B is a graph illustrating the number of bacteria sampled(cells/cm²) by ultrasound coupling with tris-HCl, swabbing, andsurfactant scrub technique measured by quantitative PCR.

FIG. 18 is a graph illustrating the effect of adding various sensitivityenhancers in LPM for enhanced detection of a model analyte human IgEantibody. Sensitivity enhancers used in the analysis are a mixture of10% NO; BSA and 0.5% w/v Tween 20 in phosphate-buffered saline (PBS)(open diamond); and a mixture of 10% w/v BSA and 0.5% w/v Tween 20 intris-buffered saline (closed circle). Prior to analysis, each of thesensitivity enhancers was diluted at 1:10 ratio with LPM containingmodel analyte. As a control, LPM containing model analyte (open square)and a commonly-used analytical solvent comprising of a mixture of 1% w/vBSA and 0.05% w/v Tween 20 in tris-buffered saline (solid square) wereused. The LPM was composed of a solution of 1% w/v mixture of NLS andBrij 30 in PBS. Error bars indicate the standard deviation.

FIGS. 19A and 19B are graphs illustrating delivery of Inulin across pigskin and delivery of Acyclovir into pig skin.

FIG. 19A shows a graph illustrating delivery after ultrasoundapplication.

FIG. 19B shows a graph illustrating delivery after abrasion with aplurality of bristles.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Energy” as used herein means any appropriate energy that can be appliedto tissue to accomplish the objective of the methods disclosed herein(e.g., liquefying tissue) Exemplary types of energy include mechanicalenergy (e.g., abrasion, shear, vacuum, pressure, suction), ultrasound,optical (e.g., laser), magnetic, thermal, and electrical energy.

An “analyte” as used herein means any biomolecule (e.g., polypeptide,nucleic acid, lipid, and the like), drug (e.g., therapeutic drugs,drugs-of-abuse, and the like), small molecule (e.g., naturalmoisturizing factors, nicotine, and the like, with the understandingthat small molecules can also be drugs), warfare agent, environmentalcontaminant (e.g., pesticides, etc.), microbe (e.g., bacterium, virus,fungus, yeast, and the like) and the like that is present in or on thetissue and can be extracted from the tissue of interest skin, a mucosalmembrane, and the like) and detected, analyzed, and/or quantified.

The term “liquefaction” is used to describe the process by which tissueand/or tissue constituents are converted to a sufficiently soluble statethrough exposure to sufficient energy and, optionally, a liquefactionpromoting medium, and can involve conversion of at least a portion of atissue structure of interest to a liquid form. A tissue sample that hasbeen subjected to liquefaction as sometimes referred to herein as a“liquefied” sample.

The term “liquefaction-promoting medium” (LPM) is used to describe asubstance that facilitates solubilization of one or more tissueconstituents, facilitates conversion of at least a portion of a tissuestructure into a liquid when exposed to energy, and/or facilitatespreservation of bioactivity of one or more solubilized tissueconstituents.

The term “liquefaction-promoting agent” (LPA) is used to describe acomponent of the liquefaction promoting medium, particularly an agentthat promotes at least solubilization and/or preservation of bioactivityof one or more tissue constituents, and/or analysis of subsequentdiagnostic assays.

A “calibration analyte” as used herein means any molecule naturallypresent in a tissue of interest at a known concentration, which canserve as a reference analyte (e.g., as a positive control to ensure adesired degree of liquefaction was achieved).

A “biomolecule” as used herein means any molecule or ion which has abiological origin or function. Non-limiting examples of biomoleculesinclude proteins (e.g., disease biomarkers such as cancer biomarkers,antibodies: IgE, IgG, IgA, IgD, or IgM, and the like), peptides, lipids(e.g., cholesterol, ceramides, or fatty acids), nucleic acids (RNA andDNA), small molecules (e.g., glucose, urea, creatine), small moleculedrugs or metabolites thereof, microbes, inorganic molecules, elements,or ions (e.g., iron, Ca2+, K+, Na+, and the like). In some embodiments,the hiomolecule is other than glucose and/or is other than a cancermarker.

The term “abused drug” or “drug-of-abuse” or “illicit drug” are usedinterchangeably herein to refer to any substance which is regulated by agovernmental (e.g. federally or state regulated) of which presence in ahuman tissue, and/or presence above a certain level in a human tissue,is illegal or can be harmful to a human being. Examples of abused drugsinclude: cocaine, heroin, methyl amphetamine, and prescription drugstaken in excess of dosage, or taken without a prescription (e.g.,painkillers, such as opioids).

The term “warfare agent” as used herein refers to any molecule,compound, or composition of either biological or chemical origin thatmay be used as a weapon. Examples of warfare agents include nerve gases(e.g. VX, Sarin), phosgene, toxins, spores (e.g., anthrax and the like.

The term “environmental contaminant” as used herein includes anymolecule, compound, or composition which can be detrimental to anindividual, e.g., when at concentrations elevated above a riskthreshold. Examples include water pollutants (e.g., fertilizers,pesticides, fungicides, insecticides, herbicides, heavy metals,halides), soil pollutants (e.g., fertilizers, pesticides, fungicides,insecticides, herbicides, heavy metals, halides), air pollutants (e.g.,NOx, SOx, greenhouse gases, persistent organic pollutants (POPs),particulate matter, smog).

The term “decontamination” as used herein includes removal from tissuesof any unwanted or undesired molecule, compound, or composition whichcan be detrimental to an individual. Examples include environmentalcontaminants (as defined above), toxic chemicals, and biological toxins.

The term “natural moisturizing factor” (NMFs) as used herein means anyone of several types of small molecules, including but not limited tofree amino acids, lactate, and urea, which are derivatives of fillagrin.NMFs can be used as analytes to facilitate assessment of general skinhealth (e.g., dry skin, flaky skin, normal skin, etc.). The term“mechanical index” as used herein means the ratio of the amplitude ofpeak negative pressure in an ultrasonic field and the square- root ofthe ultrasound frequency (Mechanical Index=(Pressure (MPa))/(Frequency(MHz)) ̂0.5.

The term “drug delivery” as used herein means the delivery of one ormore drugs into blood, lymph, interstitial fluid, a cell or tissue.

The term “sensitivity enhancer” as used herein means a substance or amixture of substances that is mixed with LPM to stabilize liquefiedtissue analytes and facilitate their analysis in terms of enhancing thesensitivity and specificity of the diagnostic analytical tests.

The term “blocking reagent” is used to describe a component which isused to prevent non-specific binding of analytes to substrates used in adiagnostic assay.

Before the present invention and specific exemplary embodiments of theinvention are described, it is to be understood that this invention isnot limited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither, or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “atissue” includes a plurality of such tissues and reference to “theliquid” includes reference to one or more liquids, and so forth. It isfurther noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike, in connection with the recitation of claim elements, or use of a“negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to he independently confirmed.

The current invention provides systems, methods and devices, as well ascompositions useful in such systems, methods and devices, involvingapplication of energy to a tissue of interest to generate a liquefiedsample comprising tissue constituents so as to provide for rapid tissuesampling, as well as qualitative and/or quantitative detection ofanalytes that may be part of tissue constituents (e.g., several types ofbiomolecules, drugs, and microbes). Determination of tissue compositionor constituents can be used in a variety of applications, includingdiagnosis or prognosis of local as well as systemic diseases, evaluatingbioavailability of therapeutics in different tissues following drugadministration, forensic detection of drugs-of-abuse, evaluating changesin the tissue microenvironment following exposure to a harmful agent,decontamination, and various other applications.

Another object of the current invention is to provide a method anddevice for liquefying a tissue of a subject for facilitating the passageof a drug across or into the tissue. The method and device disclosedabove are applicable not only to collection of tissue constituents butalso to drug delivery. The device and method involve applying energy anda liquefaction medium to a tissue of interest of a subject, anddelivering a drug through or into the site of the tissue to beliquefied. The advantage of using the present invention is 1) to providehigher fluxes of drugs into a tissue, and 2) to allow greater control offluxes into a tissue. Drugs which would simply not pass through thetissues such as the skin and into the circulatory system are forcedthrough the tissues when the method is applied.

Although the present invention may be described in conjunction withhuman applications, veterinary applications are within the contemplationand the scope of the present invention

TISSUE DIAGNOSTICS Energy Application Devices

The tissue liquefaction devices disclosed herein can be generallydescribed as having an energy source/generator operably coupled to areservoir unit/housing, where the reservoir houses a medium in whichanalytes are collected and which, in most embodiments, facilitatestransfer of energy to the tissue of interest and can thus, wheredesired, facilitate liquefaction of a tissue sample. In use, thereservoir housing is placed in contact with the subject's tissue to makecontact between the medium and the tissue, and the energy source isactivated. The device can be operably coupled to additional energysources, (e.g., abrasive actuator, piezoelectric transducer, suction orpressure), which can also be applied to the tissue to facilitatetransfer of energy to the tissue. As energy is applied to the tissue,constituents of the tissue are solubilized by the energy and collectedin the medium. The medium can be retained in the reservoir housing, oralternatively be transferred to a separate container. The reservoirhousing or container can be operably coupled to a detection device thatcan quantitatively measure the tissue constituents present in themedium.

Energy can be applied to the tissue from a single energy source or as acombination of sources. Exemplary energy sources include mechanical(e.g., abrasion, shear, vacuum, pressure, and the like), piezoelectrictransducer, ultrasound, optical (e.g., laser), thermal, and electricalenergy. The intensity of the energy applied, as well as the duration ofthe energy application, may be appropriately adjusted for the particulartissue of interest and the particular application of the method. Theenergy intensity and duration of application may also be appropriatelyadjusted based on the particular liquefaction promoting medium (LPM)used in connection with the energy. In some embodiments, an energyexposure time of greater than 1 minute, greater than 90 seconds, orgreater than 2 minutes is provided in order to produce a suitableliquefied tissue sample. The magnitude of energy depends on the analyteof interest and the selection of LPM. Higher energies are required toliquefy tissues in the absence of surfactants or particles in the LPM.Use of high energies is limited by their adverse effects on the tissueor its constituents. A significant adverse effect is injurious tissuedamage. In some embodiments, therefore, it might be necessary toincorporate certain device components that provide temporal monitoring(ideally, in real-time) of the change in tissue properties or the extentof tissue liquefaction such that, once safe limit for energy exposure isreached, the device can be stopped. The temporal evaluation can beperformed prior, during, and after liquefaction process. In certainembodiments, the temporal evaluation is performed by electrochemical(e.g., tissue's electrical conductivity, measurement of certain ions byion-selective electrodes, etc.), biochemical (e.g., measurement ofcertain tissue components in the LPM by enzymatic assays such as ELISAand the like), or optical (e.g., measurement of LPM turbidity byspectrophotometer, etc.) means. In an exemplary embodiment, tissue'stemporal electrical conductivity is measured by applying a pre-definedAC electrical voltage across the tissue with a signal generator, andanalyzing the resultant electrical current by a multimeter. Anothersignificant adverse effect of high energy exposure is attributed totemperature elevation in the tissue, also known as thermal effects. Insome embodiments, therefore, it might be necessary to incorporate atemperature sensing element (e.g., a thermocouple) that allowsmonitoring of the temperature of the tissue and/or the LPM, facilitatingthe judgment of a safe amount of energy exposure to the tissue.

The necessary energy level is significantly reduced by appropriateselection of LPM. For example, use of saline alone along with ultrasoundresulted in recovery of less than 0.1 mg protein per cm² of skin. On theother hand, incorporation of surfactants such as DPS, NTS and Brij-30 ata concentration of 1% w/v in LPM increased protein recovery to more than0.6 mg per cm² of skin.

In certain embodiments, use of energy to liquefy tissue may lead toreduction in biological activity of solubilized tissue constituents,necessitating selection of LPM which adequately preserve the bioactivityof tissue's molecules as well as aid tissue solubilization. For example,incorporation of one or more surfactants such as DPS, NLS and Brij-30 ata concentration of 1% w/v in LPM facilitated complete preservation ofthe bioactivity of solubilized proteins and nucleic acids underultrasonic energy exposure.

In certain embodiments, energy can be applied to a tissue using anenergy delivery chamber that includes an energy producing element. Thechamber, when placed on the tissue, will expose the tissue to the energyproducing element and allow energy to be applied to the tissue withminimal interference, Such a chamber can contain LPM and provide forcontact of the LPM with the tissue such that, upon application ofenergy, tissue constituents can be directly collected into the solution.

In certain embodiments, the energy delivery chamber containing the LPMmay also comprise a diagnostic device, for example, an analyte sensor,for detecting and, optionally, quantifying analytes that may be presentin the LPM. These diagnostic devices can serve as chemical sensors,biosensors, or can provide other measurements to form a completesampling and measurement system. An element having an internal channelfor fluid transfer can be fabricated together with a sensor to form adisposable unit. The device can also be adapted to include or beprovided as a disposable unit that provides for collection of analytesin the LPM for analysis.

Alternatively, the diagnostic element can be located elsewhere (e.g.,separate from the energy device) and the contents of the energy deliverychamber in contact with tissue can be pumped using mechanical forces,capillary forces, ultrasound, vacuum, or electroosmotic forces into asensing chamber and analyzed.

In certain embodiments, e.g., when evaluating topical formulations ordetermining pharmacological parameters, the unit can be constructed tofunction as a closed loop drug delivery unit, including drug deliverymeans, analyte recovery means, sensing means to measure the analyte, andcontrol means to provide a signal to the drug delivery means.

An example of the general operation of an energy-assisted analyte deviceis described here. A portable disposable unit is inserted into aportable or bench-top energy generator. The energy generator may alsoinclude circuitry for tissue resistance measurements, analyteconcentration measurements, and display of analyte concentrationmeasurements. The system (e.g., energy applicator and disposable unit)is placed against the tissue, and energy is applied for a certain periodof time, either alone or as a combination with other physical,mechanical, electrical, and chemical forces. The tissue of interest isliquefied, and analytes from the liquefied tissue are collected in thedisposable unit and are measured using appropriate assays.

The preferred embodiment of the present invention and its advantages arebest understood by referring to FIGS. 1 through 19 of the drawings, likenumerals being used for like and corresponding parts of the variousdrawings.

Referring to FIGS. 1A through 1G, the structure, components andfunctioning of abrasive energy-based tissue liquefaction devices areshown. FIGS. 1A through 1C show the sequential working of a device thatutilizes a rotary abrasive component 101 as means for applying energy totissues for liquefaction. Liquefaction is achieved by placing andsetting abrasive component 101 in motion against a tissue of interest107. Abrasive component 101 is attached to a shaft 102, which is furtherconnected to a rotary motor 103 in the device, In some embodiments,shaft 102 is designed to sense and control the pressure applied byabrasive component 101 on tissue 107. In an exemplary embodiment, shaft102 is constructed of shaft 1021 and shaft 1022 which are connected toeach other by a pressure-sensitive spring 1023 (FIG. 1D). In anotherembodiment, shaft 1021 and shaft 1022 sandwich between them apressure-sensing piezoelectric crystal for monitoring and controllingapplied pressure to tissue 107. A battery pack 104 powers motor 103,which can subsequently set abrasive component 101 in rotary motion whendirected by the device operator. Prior to liquefaction, abrasivecomponent 101 is designed to be held in isolation against tissue 107using a housing 105, and specifically, a thin sheet 106 located on thebase of housing 105 (FIG. 1A). Upon initiation of the liquefactionprocess. LPM stored in a cartridge 108 is transferred to the housing 105(FIG. 1A), whereupon the LPM contacts the surface of the sheet material106, followed by setting the abrasive component 101 in motion againstsheet 106. Material of sheet 106 is chosen such that it can be quicklyabraded by abrasive component 101, allowing LPM and abrasive component101 to come in contact with tissue 107 leading to tissue liquefaction(FIG. 1B). Non-limiting examples of sheet 106 include sheet of paper,rubber sheet, metal foil, plastic sheet, or any water-soluble sheet.Upon completion of liquefaction process, motor 103 stops and LPMcontaining tissue constituents is transferred to a sample container 110(FIG. 1C) or directly into a pre-vacuumized container (thus avoiding theneed for suction pump 109 and container 110). Where there is nopre-vacuumized container, collection of the sample is facilitated by asuction pump 109.

In some embodiments, certain device components are designed asdisposable units such that, after each use of the device, thesecomponents can be replaced to allow sterile usage. Such components mayinclude housing 105, abrasive component 101, cartridge 108, samplecontainer 110, and other fluid-handling device components, as deemednecessary to maintain device sterility. Alternatively, in someembodiments, the whole device may be made disposable.

In certain embodiments, LPM storing cartridge 108 can be replaced with asponge-bellow assembly for storage and release of LPM. FIGS. 1E through1G show the sequential working of such a device. A flexiblebellow-shaped housing 112 contains a sponge 111 filled with LPM (FIG.1E). As the device is pushed against tissue 107, sponge-bellow housingis squeezed to release LPM and abrasive component 101 is set in motion(FIG. 1F). Upon completion of liquefaction process, motor 103 stops andLPM containing tissue constituents is transferred to a sample container110 (FIG. 1G). Collection of the sample is facilitated by a suction pump109. Alternatively, in some embodiments the suction pump 109 andcontainer 110 may be avoided by collecting the sample into the sponge bylifting the device back into its original position.

Referring to FIGS. 2A and 2B, the structure and components of moveabletissue liquefaction devices designed for continuous sampling of a largearea of tissue are shown. FIG. 2A shows a device that utilizes a rotaryabrasive component 201 as means for applying energy to tissues forliquefaction. Liquefaction is achieved by placing and setting abrasivecomponent 201 in motion against a tissue of interest 207. Abrasivecomponent 201 is attached to a shaft 202, which is further connected toa rotary motor 203 in the device. In some embodiments, shaft 202 isdesigned to sense and control the pressure applied by abrasive component201 on tissue 207. In an exemplary embodiment, shaft 202 is constructedof two distinct shafts which are connected to each other by apressure-sensitive spring or a pressure-sensing piezoelectric crystalfor monitoring and controlling the applied pressure to tissue 207. Abattery pack 204 powers motor 203, which can subsequently set abrasivecomponent 201 in rotary motion when directed by the device operator.Once the device is placed against tissue 207, a continuous liquefactionprocedure is initiated by performing three key processes—LPM stored in acartridge 208 is continuously delivered to housing 212 at thedevice-tissue interface; abrasive component 201 is set in motion againsttissue 207; and liquefied tissue sample is continuously collected in asample container 210 using a suction pump 209. The device can be movedaround such that additional tissue surfaces are exposed to the deviceand liquefied. When desired, the liquefaction process can be stopped byswitching-off motor 103 and cumulative tissue sample can be accessedfrom container 210.

In some embodiments, additional device components may be used forpreventing LPM leakage from housing 212 due to the motion of device overtissue surface. In an exemplary embodiment, suction pump 209 can be usedto create a vacuum-assisted seal between tissue 207 and chamber 206located in a flanged housing 205 around the device.

FIG. 2B shows a device that utilizes a piezoelectric element 251 asmeans for applying mechanical energy to tissues for liquefaction.Piezoelectric element 251 is placed in a housing 252 that interfaceswith a tissue of interest 259, and liquefaction is achieved byactivating piezoelectric element 251 with LPM present as a couplingfluid between tissue 259 and piezoelectric element 251. Piezoelectricelement 251 is a transducer of electrical energy, which is supplied toit by means of circuitry placed in a flexible tubing 253, Duringliquefaction, LPM is supplied to housing 252 by a flexible tubing 254using an operator-controlled injection system 256. Liquefied tissuesample can be simultaneously collected from housing 252 into a samplecontainer 257 using a flexible tubing 255. Sample collection isfacilitated by a suction pump 258 which is serially connected to samplecontainer 257. In some embodiments, suction pressure created in housing252 by suction pump 258 may provide for an effective seal betweenhousing 252 and tissue 259 for preventing LPM leakage from housing 252during liquefaction. In some embodiments, suction pressure created inhousing 252 by suction pump 258 may provide for an additional source ofenergy for liquefaction.

In some embodiments, housing 252 may be moved to liquefy additionaltissue surfaces and collect a sample representing tissue constituentsaccumulated from various tissue surfaces. in such a device LPM iscontinuously supplied to housing 252 by tubing 254 and sample iscontinuously collected by tubing 255.

In certain embodiments, the device in FIG. 2B may operate without apiezoelectric element 251. In this embodiment, the LPM which flows froma tubing 254 into the housing 252 makes contact with the tissue andliquefies the tissue. Liquefied tissue is collected from the housing bytubing 255. The housing may be moved continuously or intermittently tocollect samples from a large tissue area. The device may have additionalmeans that are practically necessary to allow the movement of the deviceon a tissue, liquefaction of tissue and collection of liquefied tissue.In certain embodiments, either pressure or vacuum but not both may beused to direct LPM towards the tissue and collect liquefied tissue.

In certain embodiments, liquefaction devices may be integrated with adiagnostic probe such as endoscope, colonoscope, laparoscope, and thelike.

Referring to FIGS. 3A through 3E, the structure and components ofliquefaction devices that utilize an oscillating abrasive component asmeans for applying energy to tissues for liquefaction are shown.Referring to FIGS. 3A and 3B, liquefaction is achieved by placing andsetting abrasive component 301 in motion against a tissue of interest311. Linear motion can be achieved, for example, by a rack and pinionarrangement (FIGS. 3A and 3B). Specifically, abrasive component 301 isattached to a rack 302, which slides in a linear oscillatory motionusing a circular gear 303 (pinion). Gear 303 is driven in oscillatorycircular motion by a motor 304. A battery pack 305 powers motor 304. insome embodiments, motor 304 is a servo motor which may require anelectronic microchip controller 306 to produce oscillatory circularmotion. Prior to liquefaction, abrasive component 301 is designed to beheld in isolation against tissue 311 using a housing 307, andspecifically, a thin sheet 308 located on the base of housing 307. LPMcan be pre-stored in housing 307, for instance, so that it is in contactwith 308. In some embodiments, LPM may be transferred to housing 307from a cartridge located elsewhere in the device. Liquefaction processis initiated by setting the abrasive component 301 in linear motionagainst sheet 308. Material of sheet 308 is chosen such that it can bequickly abraded by abrasive component 301, allowing LPM and abrasivecomponent 301 to come in contact with tissue 311 leading to tissueliquefaction. Non-limiting examples of sheet 311 include sheet of paper,rubber sheet, metal foil, plastic sheet, or any water-soluble sheet.Upon completion of liquefaction process, motor 304 stops and LPMcontaining tissue constituents is transferred to a sample container 309.Collection of the sample is facilitated by a suction pump 310. Incertain embodiments, the sample may be directly collected in apre-vacuumized container, avoiding the need of suction pump 310 andcontainer 310.

In some embodiments, certain device components are designed asdisposable units such that, after each use of the device, thesecomponents can be replaced to allow sterile usage. Such components mayinclude housing 307, abrasive component 301, sample container 309, andother fluid-handling device components, as deemed necessary to maintaindevice sterility. Alternatively, in some embodiments, the whole devicemay be made disposable.

In some embodiments, the linear oscillatory motion of abrasive component301 may be generated by other mechanism such as using linear motors,linear motion actuators, ball screw assembly, leadscrew assembly,jackscrew assembly, and other devices for translating rotational motionto linear motion.

In some embodiments, a single rack and pinion system as described inFIGS. 3A and 3B may be replaced with an arrangement of multiple gearsand a belt as exemplified in FIGS. 3C and 3D. Specifically, a belt 327is mounted on gears 321, 322, 323, 324, 325 and 326. An abrasivecomponent 328 is attached to belt 327 and is set in a linear oscillatorymotion when gear 321 is driven by motor 304 in an oscillatory rotationmotion. While gears 321, 322 and 326 are fixed to the housing of device,gears 323, 324 and 325 are mounted on shaft 328. Shaft 328 is fixed tothe housing of device. In some embodiments, shaft 328 has a flexiblelength such that, as abrasive component 328 is pressed against anon-flat tissue surface, shafts 328 attached with gears 323, 324 and 325are able to adjust their lengths in order to make abrasive component 328contour with the non-flat tissue surface. Additionally, shaft 328 may bedesigned to sense and control the pressure applied by abrasive component328 on tissue surface, In an exemplary embodiment, shaft 328 isconstructed of shaft 3281 and shaft 3282 which are connected to eachother by a pressure-sensitive spring 3283 (FIG. 3E).

Referring to FIGS. 4A through 4G, several designs of abrasive componentused in devices, methods and systems disclosed in this invention aredescribed. FIG. 4A illustrates an abrasive component comprising of asheet of abrasive material with uniform thickness. Non- limitingexamples of abrasive material with uniform thickness include fabric,abrasive crystals (e.g., quartz, metal, silica, silicon carbide, dustand derivatives of aluminum (such as AlO₂), diamond dust, polymeric andnatural sponge, and the like, etc. In some embodiments, it may beadvantageous to design an abrasive component with heterogeneousabrasiveness, for example, those having spatial variation ofabrasiveness. in an exemplary embodiment, abrasive component is a discwith a gradient of abrasiveness that varies from high abrasiveness atdisc's center to low abrasiveness at the disc periphery (FIG. 4B). Insome embodiments, the shape of abrasive component may be varied to anon-planar geometry. In exemplary embodiments, FIG. 4C shows an abrasivecomponent with a smooth and rounded tissue-facing surface (aspect ratiodefined as the ratio of height and width—may vary from 10 to 0.1), andFIG. 4D shows a circular ring-shaped abrasive component. FIGS. 4Ethrough 4G show embodiments of abrasive components using brush as meansfor tissue abrasion. FIG. 4E illustrates an abrasive componentcomprising of a brush with bristles of uniform height and abrasiveness.In some embodiments, abrasive component comprises of a brush withbristles of different height and/or abrasiveness. FIG. 4F shows anexemplary embodiment of a circular disc-shaped brush with bristles ofhigh abrasiveness at the center surrounded by bristles with lowabrasiveness in the disc periphery. FIG. 4G shows an exemplaryembodiment of a brush with bristles of different lengths forming asmooth and rounded tissue-facing surface (aspect ratio defined as theratio of height and width of abrasive component—may vary from 10 to0.1).

Referring to FIGS. 5A through 5D, device components for measuring atissue's electrical conductivity are disclosed. While high energyexposure favorably liquefies tissues, its use may lead to significantadverse effects such as injurious tissue damage. In some embodiments,therefore, it might be necessary to incorporate certain devicecomponents that provide temporal monitoring (ideally, in real-time) ofthe change in tissue properties, e.g., tissue's electrical conductivity,such that, once safe limit for energy exposure is reached, the devicecan be stopped. Temporal measurement and monitoring of tissue'selectrical conductivity during liquefaction process can be done byapplying a pre-defined AC electrical voltage across the tissue ofinterest 503 using a measurement electrode 501 placed on tissue 503 anda reference electrode 502 placed in the vicinity of the region on tissue503 that is being liquefied. The resultant electrical current across thetwo electrodes, as measured by an ammeter 504, can be taken as a measureof tissue's electrical conductivity. In some embodiments, measurementelectrode 501 is maintained in electrical contact with LPM, or directlywith the region on tissue 503 that is being liquefied. In an embodiment,measurement electrode 501 is located as an inner surface lining of LPMhousing 509 (FIG. 5A). In certain embodiments, measurement electrode isa sliding contact 506 that is fastened to a motorized shaft 510 immersedin (FIG. 5B). Electrical current is transmitted by sliding contact 506to an isolated stud 505 secured on the device housing. In someembodiments, reference electrode 502 is an extension of LPM housing 509and is placed in peripheral vicinity of the region on tissue 503 that isbeing liquefied (FIG. 5A and FIG. 5B). In some embodiments, referenceelectrode is a handheld cylindrical electrode 507 that is electricallyconnected with the electrical conductivity measurement componentslocated in the liquefaction device (FIG. 5C). in some embodiments,reference electrode is a patch electrode 508 that is electricallyconnected with the electrical conductivity measurement componentslocated in the liquefaction device (FIG. 5D).

Referring to FIGS. 6A through 6J, structure, components and functioningof devices utilizing microneedle-based tissue liquefaction aredisclosed. Microneedle-based devices apply energy to tissues throughmechanical disruption of tissue components which is primarilyaccomplished by pushing microneedles into the tissue, FIG. 6A shows thebasic design of a microneedle patch 601 bearing a multitude ofmicroneedles 602 which are pre-filled with LPM 613. Microneedle patch601 can be inserted in the tissue of interest allowing disruption anddissolution of tissue components in LPM 613. LPM 613 can be lateraspirated from patch 601 for diagnostic analysis.

Additional energy for liquefaction may be applied by post-insertionmotion of microneedles inside the tissue. FIG. 6B illustrates avibratory component 603 which may be secured on microneedle patch 601,which after insertion of patch 601 into tissue can be activated tovigorously shake microneedles 602 inside the tissue. Vibratory component603 contains a multitude of mechanical vibrators 6031 and abattery-operated electronic circuit board 6032 for powering andcontrolling the motion of mechanical vibrators in desired directions. Inan exemplary embodiment, mechanical vibrators 6031 can be vibrated indirections parallel and perpendicular to the axis of microneedles 602.

In some embodiments, motion of microneedles post-insertion may beproduced by the motion of each microneedle 602 with respect to patch601. FIGS. 6G and 6H discloses an electromagnet 612 placed on top ofpatch 601. Electromagnet 612 may be used to produce oscillatory motionof each microneedle 602 along its axis. This can be achieved byfastening a magnet 611 on top of each microneedle 602, such that magnet611 responds to an alternating polarity profile of electromagnet 612leading to oscillatory linear motion of microneedles 602. In certainembodiments rotary motion of microneedles may be desired. Electromagnets6121, 6122, 6123 and 6124 are placed symmetrically around patch 601(FIGS. 6I and 6J). Magnet 611 attached on top of each microneedle 602responds to alternating polarity profile of electromagnet 6121, 6122,6123 and 6124 leading to rotary motion of microneedles 602.

In FIGS. 6B through 6F, additional energy for liquefaction may befurther applied by forced motion of LPM in tissue using active injectionand withdrawal of LPM through microneedles. A housing 604 placed in thedevice may contain a compressed air container 605 which can be utilizedto force LPM contained in patch 601 to flow inside tissue. A suctionpump 606 in housing 604 may be used to apply vacuum for withdrawing LPMfrom tissue, in some embodiments, compressed air container 605 andsuction pump 606 may be alternatively used for repeated injection andwithdrawal of LPM from tissue for enhanced liquefaction. Abattery-operated electronic circuit board 607 in housing 604 is used forpowering and controlling compressed air container 605 and suction pump606. In some embodiments, suction pump 606 may be additionally connectedto a sample container to aspirate and transfer liquefied tissue samplefrom patch 601 to the sample container. In certain embodiments, housing604 may be replaced by a flexible elastic cap 608 (see FIGS. 6D and 6E)fitted on top of patch 601. Flexible cap 608 may be repeated pushed inand pushed out, for example, by pushing with a finger, such that LPM isrepeatedly injected and withdrawn from the tissue through microneedles602.

Microneedles 602 may be coated with a substance 610 to enhance tissueliquefaction (FIG. 6F). In some embodiments, substance 610 is anabrasive material which may help in enhanced disruption of tissueconstituents and their faster dissolution in LPM. In some embodiments,substance 610 is an enzyme which may cleave specific tissue componentssuch as extracellular matrix for enhanced tissue liquefaction. In someembodiments, substance 610 is a molecule that specifically binds totissue analytes of interest leading to enhanced recovery of the analytefrom the tissue. In an exemplary embodiment, substance 610 is anantibody.

Referring to FIG. 6A through 6F, in some embodiments, certain devicecomponents may be designed as disposable such that, after each use ofthe device, these components can be replaced to allow sterile usage.Such components may include microneedle patch 601, microneedles 602,compressed air container 605, suction pump 606 and other fluid-handlingdevice components, as deemed necessary to maintain device sterility.Alternatively, in some embodiments, the whole device may be madedisposable.

Liquefaction-Promoting Medium (LPM)

The LPM can be designed to serve one or more of the following fourpurposes: a) it facilitates dispersion of tissues into its constituents,b) it acts as a medium to collect liquefied tissue constituents, and c)it inhibits degradation of the sampled constituents such that theirchemical or biological activity is retained (e.g., by preserving variousmolecules' structural conformation and by preserving the ability ofsampled microbes to multiply), and d) ensure compatibility to thesubsequent analytical techniques.

In general, LPM comprises a solvent, such as aqueous solutions (e.g.,tris-HCl, phosphate buffered saline, etc.) or organic (“non-aqueous”)liquids (e.g., DMSO, ethanol, and the like), which may additionallycontain a variety of liquefaction-promoting agents, including but notlimited to surfactants (non-ionic, anionic, or cationic), fatty acids,azone-like molecules, chelating agents (e.g., EDTA, etc.), inorganiccompounds, and abrasive substances. “Liquefaction-promoting agent” asused herein refers to a component of a LPM which can facilitateliquefaction of a tissue sample and/or solubilization of tissueconstituents. Depending on the tissue type and the analytes of interest,constituents of the LPM can be rationally selected based on the criteriadescribed above. For example, a delicate tissue, such as mucosalmembrane, can be liquefied by a saline solution with minimal or nosurfactants, whereas keratinized tissues, such as skin, will requireadditional constituents, such as surfactants.

The liquefaction promoting agents within the LPM can comprise a varietyof suitable components including, but not limited to: water, tris-HCl,saline (phosphate-buffered saline (PBS), and tris-buffered saline(TBS)), alcohols (including ethanol and isopropanol (e.g., in aconcentration range of 10-100% in aqueous solution)), abrasivesubstances, such as dust or derivatives of silica, aluminum oxide, orsilicon carbide (e.g., in a concentration range of 0.01-99% (w/v) inwater-based solution), surfactants, such as Brij (various chain lengths,e.g., Brij-30), 3-(Decyl dimethyl ammonio) propane sulfonate (DPS),3-(Dodecyl dimethyl ammonio) propane sulfonate (DDPS),N-lauroylsarcosine (NLS), Triton X-100, sodium dodecyl sulfate (SDS) and sodiumlauryl sulfate (SLS), HCO-60 surfactant, hydroxypolyethoxydodecane,lauroyl sarcosine, nonoxynol, octoxynol, phenylsulfonate, pluronic,polyoleates, sodium laurate, sodium oleate, sorbitan dilaurate, sorbitandioleate, sorbitan monolaurate, sorbitan monooleates, sorbitantrilaurate, sorbitan trioleate, Span 20, Span 40, Span 85, SynperonicNP, Tweens, sodium alkyl sulfates, and alkyl ammonium halides, (e.g., inconcentrations ranging between 0.01-20% in water-based solution), DMSO(e.g., in a concentration range of between 0.01-20% in water-basedsolution), fatty acids such as linoleic acid (e.g., in a concentrationrange of between 0.1-2% in ethanol:water (50:50), azone (e.g., in aconcentration range of 0.1-10% in ethanol:water (50:50), polyethyleneglycol (e.g., in a concentration range of 10-50% in water-basedsolution), histamine (e.g., in a concentration range of 10-100 mg/ml inwater-based solution), EDTA (e.g., in a concentration range of 1-100mM), and sodium hydroxide (e.g., in a concentration range of 1-100 mM).In some embodiments the LPM may contain surfactants other than TWEEN,CTAB, SPAN, or sodium alkyl sulfate. In some embodiments, the LPM maycontain surfactants other than cationic surfactants. Where the LPMincludes a surfactant, the total concentration of the surfactant (w/v inthe LPM can range from at least 0.5%, to 10%, and can be, for example,about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, or about 3%.

The LPM can include agents that facilitate preservation of bioactivityof an analyte of interest. For example, the LPM can contain free radicalscavengers (e.g., antioxidants (e.g., polyphenol, beta-carotene, lutein,lycopene, selenium, etc.), vitamin A. vitamin C, vitamin E.alpha-tocopherol, butylated hydroxytoluene, sodium benzoate, sodiumformate, and the like); defoaming agents (e.g., silicone or non-siliconeanti-foaming agents such as dimethylpolysiloxane, hydrocarbon oil, lowfatty acid diglyceride, and the like); and shear protectants (e.g.,polyethylene glycol, polyvinyl alcohol, plutonic F68, and the like),“Bioactivity” as used in the context of an analyte refers to astructural conformation that facilitates detection (e.g., such as anepitope bound by a specific antibody or other structural feature that issensitive to denaturation), and may also include a biological activityof an analyte (e.g., enzymatic activity).

LPM of particular interest are those that contain a combination ofsurfactants that when used in connection with the devices, methods andsystems disclosed herein provides for a desired level of tissueconstituents in the LPM while providing for preservation of bioactivityof analytes in the LPM, particularly so as to provide for maintenance ofstructural conformation of an analyte (e.g., avoid denaturation of aprotein analyte).

Use of different combinations of surfactants including combination ofnonionic surfactant, zwitterionic surfactant and anionic surfactant inthe LPM may provide for both high levels of tissue constituents in theLPM and good preservation of bioactivity of an analyte contained in theLPM following use in devices, methods and systems described herein.

Non-limiting examples of non-ionic surfactants of interest include Brijseries surfactants (e.g., polyethylene glycol dodecyl ether (Brij 30),polyoxyethylene 23-lauryl ether (Brij 35), polyoxyethylene 2-cetyl ether(Brij 52), polyoxyethylene 10-cetyl ether (Brij 56), polyoxyethylene20-cetyl ether (Brij 58), polyoxyethylene 2-stearyl ether (Brij 72),polyoxyethylene 10-stearyl ether (Brij 76), polyoxyethylene 20-stearylether (Brij 78), polyoxyethylene 2-oleyl ether (Brij 92),polyoxyethylene 10-oleyl ether (Brij 96), polyoxyethylene 100-stearylether (Brij 700), polyoxyethylene 21-stearvi ether (Brij 721), and thelike); Triton X (e.g., Triton X-15, Triton X-45, Triton X-100, TritonX-114, Triton X-165, Triton X-200, Triton X-207, Triton X-305, TritonX-405, and the like); and Sorbitan (e.g., Span-20, Span-40, Span-60,Span-65, Span-80, Span-85, and the like).

Non-limiting examples of zwitterionic surfactants of interest include3-(decyl dimethyl ammonio) propane sulfonate, 3-(dodecyl dimethylammonio) propane sulfonate, myristyldimethyl ammonio propane sulfonate,hexadecyldimethyl ammonio propane sulfonate, ChemBetaine C, ChemBetaineOleyl, ChemBetaine CAS, and 3 -(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate.

Non-limiting examples of anionic surfactants of interest includeN-lauroyl sarcosine, sodium cocoyl sarcosinate, sodium myristoylsarcosinate, isopropyl lauroyl sarcosinate, sodium palmitoylsarcosinate, and disodium lauroaphodiacetate lauroyl sarcosinate.

In some embodiments, non-ionic surfactants are combined withzwitterionic surfactants. In certain embodiments, non-ionic surfactantsare combined with anionic surfactants. In these embodiments, the ratioof non-ionic surfactant to zwitterionic, or anionic surfactant presentin the LPM can be adjusted to achieve desired results. Non-limitingratios of interest include 25:75 non-ionic: zwitterionic surfactant,50:50 non-ionic: zwitterionic surfactant, 75:25 non-ionic: zwitterionicsurfactant, 25:75 non-ionic:anionic surfactant, 50:50 non-ionic:anionicsurfactant, and 75:25 non-ionic:a.nionic surfactant. A mixture ofparticular interest is a 50:50 surfactant mixture of a Brij seriessurfactant (e.g., Brij-30) and N-lauroyl sarcosine (NLS). Anothermixture of particular interest is a 50:50 surfactant mixture of a Brijseries surfactant (e.g., Brij-30) and 3-(I)ecyl dimethyl ammonio)propane sulfonate (DPS). As illustrated in the Examples below, thesecombinations of surfactants, when included in the LPM at a totalsurfactant concentration of 0.5-1% (w/v), provided for solubilization ofa high level of tissue constituents as assessed by total proteinconcentration, and provided for retention of bioactivity (as assessed byELBA technique).

In some specific cases, for example, the collection of live pathogens,different LPM compositions can be used to achieve desired results.Saline and tris-HCl were used as an LPM to provide for collection of awide variety of skin-resident bacteria, and additionally, these microbesremained potent to multiply and grow ex vivo. In some embodiments, anLPM may contain an enrichment broth medium to supports growth of sampledmicrobes. Some of anaerobic bacteria are sensitive to an oxygenatmosphere. Thus, the LPM for collecting anaerobic bacteria may containa nitrogen and hydrogen atmosphere. It will be evident to the ordinarilyskilled artisan upon reading the present disclosure that LPMcompositions varying in components can be readily produced for use inspecific applications.

LPM can also include stabilizers of analytes of interest, such asprotease-inhibitors, RNase-inhibitors, and DNase-inhibitors, which canprovide for collection and at least temporary storage of analytes withminimal or no detectable degradation or loss of bioactivity. Otherexemplary liquefaction-promoting agents are described in U.S. Pat. No.5,947,921, which is incorporated herein by reference in its entirety.For example, the liquefaction-promoting agent can include surfactants,abrasive particles, and biomolecule stabilizers.

In one exemplary embodiment, the LPM is composed of a solution of 1% w/vmixture of NLS and Brij-30 in sterile PBS. In another exemplaryembodiment, the LPM is composed of a solution of 0.5% w/v mixture of DPSand Brij-30 in sterile PBS. In certain embodiments, specifically wherethe analytes are one or more proteins, the LPM contains a 1-10% v/vprotease inhibitor cocktail (e.g., catalog number: P8340, provided bySigma-Aldrich, St. Louis, Mo.). In certain embodiments, the LPM is asaline solution. In certain embodiments, the LPM is a tris-HCl solution.

LPM can also include agents defined as “sensitivity enhancers”, whichare used to stabilize liquefied tissue analytes and facilitate theiranalysis in terms of enhancing the sensitivity and specificity of thediagnostic analytical tests. As deemed necessary to achieve these goals,the sensitivity enhancers can be added into LPM prior, during or aftertissue liquefaction process, or prior or during the diagnostic analysis.For example, the sensitivity enhancer may be pre-stored in a container,and later the liquefied tissue sample may be mixed.

In typical embodiments, sensitivity enhancers are formulated ofsubstances that synergistically act with specific components of LPM (asdisclosed above) to enhance the detection sensitivity and specificity ofanalytes of interest. In an exemplary embodiment, sensitivity enhancersare formulated of substances for preventing non-specific binding ofprotein analytes present in tissue sample to various diagnostic assaysubstrates, resulting in their sensitive and specific detection. In someembodiments, sensitivity enhancers are formulated to stabilize analytesof interest by deactivating molecules such as protease, RNase and DNase.In some specific cases, sensitivity enhancers may be formulated ofsubstances that activate proteases to prevent non-specific biding ofcertain analytes of interest with proteins present in the liquefiedsample. In some embodiments, sensitivity enhancers are used to adjustthe physiological state (for example, pH) of the liquefied samples tofacilitate downstream analysis of analytes of interest.

In some embodiments, sensitivity enhancer may comprise of a solvent,such as aqueous solutions (e.g., phosphate buffered saline,tris-buffered saline, etc.) or organic liquids (“non-aqueous”) liquids(e.g., DMSO, ethanol, phenol and the like), which may additionallycontain but not limited to blocking reagents (e.g., Tween 20, TritonX-100, bovine serum albumin, non-fat dry milk, casein, caseinate, fishgelatin, sonicated-sperm-nucleic acids and the like), stabilizers suchas protease, protease-inhibitors, RNase-inhibitors and DNase-inhibitors,broth mediums. Depending on the type of tissue and analyte of interest,components of the sensitivity enhancer can be rationally chosen. In anexemplary embodiment, for detecting nucleic acids in liquefiedkeratinized tissue such as skin, the sensitivity enhancer comprises of100 mM NaCl, 10 mM tris-HCl (pH 8), 25 mM EDTA (pH 8), 0.5% SDS, and 0.1mg/ml protease K. Herein, Protease K may not only facilitatesliquefaction of the skin but may also stabilize nucleic acids bydecomposing DNase and RNase present in the sample as a tissue analyte.

In some embodiments involving analyte detection by an immunoassay,sensitivity enhancer may comprise of a variety of suitable componentsincluding, but not limited to: solvent (e.g., water, a buffer solution(e.g., phosphate-buffered saline, tris-HCl, tris-buffered saline, etc.),and the like), a stabilizer such as a protease inhibitor, and a blockingreagent such as Tween 20, Triton X-100, bovine serum albumin (e.g., in aconcentration range of 1-5%), non-fat dry milk (e.g., in a concentrationrange of 0.1-0.5N, casein or caseinate (e.g., in a concentration rangeof 1-5%), fish gelatin (e.g., in a concentration range of 1-5%). In anexemplary embodiment, the sensitivity enhancer for immunoassays iscomposed of a solution of 10% BSA and 0.5% Tween 20 in Tris-bufferedsaline and is mixed with the tissue sample at ratio of 1:10.

In some embodiments involving detection of nucleic acids as an analyteof interest, sensitivity enhancer may comprise of various suitablecomponents including, but not limited to: water, a buffer solution(e.g., TE, TAE, sodium citrate, etc.), a chelating agents such as EDTA,a stabilizer (e.g., RNase-inhibitor, DNase-inhibitor, protease, phenol,ammonium sulfate, guanidine isothiocyanate, etc.), a surfactant such assodium dodecyl sulfate, and blocking reagents such assonicated-sperm-nucleic acids, Tween 20, Triton X-100, bovine serumalbumin (e.g., in a concentration range of 1-5%), non-fat dry milk(e.g., in a concentration range of 0.1-0.5%), casein or caseinate (e.g.,in a concentration range of 1-5%), fish gelatin (e.g., in aconcentration range of 1-5%). In some embodiments, where detection ofnucleic acids is desired by using polymerase-chain-reaction (PCR)technology, the LPM has to be chosen so as to avoid inclusion ofPCR-inhibitors as LPA. In exemplary embodiments, PCR-compatible istris-HCl buffer, or EDTA buffer.

In some embodiments involving detection microbes as an analyte ofinterest, sensitivity enhancer may comprise an enrichment broth mediumso as to facilitate growth of microbes ex vivo. Some of anaerobicbacteria are sensitive to an oxygen atmosphere. Thus, the sensitivityenhancer for collecting anaerobic bacteria may contain a nitrogen andhydrogen atmosphere.

Other formulations of sensitivity enhancer for specific assay system orspecific analyte of interest will be evident to the ordinarily skilledartisan upon reading the present disclosure.

In some embodiments, the thermal properties (e.g., temperature,heat-capacity, and the like) of the LPM can be manipulated before orduring tissue liquefaction so as to reduce the adverse thermal effectsof energy exposure on tissue and/or its constituents. In one embodiment,the temperature of the LPM is maintained low enough not to inducemelting of the tissue constituents. In another exemplary embodiment, apre-cooled LPM having temperature lower than the ambient temperature(about 25° C.) can be used for ultrasound liquefaction. In anotherexemplary embodiment, the temperature of the LPM can be continuouslyreduced during energy exposure by transferring its heat to a pre-cooledliquid flowing through a heat-transfer jacket coupled to theLPM-containing reservoir.

Analytes

A variety of analytes can be detected (qualitatively or quantitatively)with the devices, methods and systems disclosed herein and, optionally,characterized to provide an analyte profile of the tissue in question.Non-limiting examples include: structural and signaling proteins (e.g.,keratins (e.g., basic keratins, acidic keratins), -actin, interleukins,chemokines, growth factors, colony-stimulating factors, interferons,antibodies (IgE, IgA, IgD, IgM), cancer biomarkers (e.g., CEA, and thelike), heat shock proteins (e.g., Hsp-60, Hsp-70, Hsp-90, etc.), and thelike, lipids (e.g., cholesterol), ceramides (e.g., ceramides 1-6), fattyacids, triglycerides, paraffin hydrocarbons, squalene, cholesterylesters, cholesteryl diesters, free fatty acids, lanosterol, cholesterol,polar lipids (e.g., glucosyl-derivatives and phospholipids), and thelike, nucleic acids (e.g., RNA and DNA), small molecules (e.g., freeamino acids, lactate, exogenously delivered drug molecules,environmental contaminants, warfare agents, and the like) andmicroorganisms (e.g. bacteria, fungi, viruses and the like). Theseanalytes are found within the tissue itself, and may not be solelypresent in the interstitial fluid around the tissue. The analyte may beother than a marker associated with interstitial fluid, such as a tumormarker. Thus, the devices, methods and systems disclosed herein can beadapted to detect tumor markers that are present in tissue structures,but which may or may not also be present in interstitial fluid.

In a particular embodiment, antibodies against allergens and cytokinesare liquefied (are these liquefied or is the tissue liquefied to producethese soluble analytes) and characterized to provide an allergy profilefor the tissue and the subject in question. Specific types of antibodiesinclude but are not limited to IgE and IgG antibodies. Specific types ofcytokines include but are not limited to IL4, IL5, IL10, IL-12, IL13,IL-16, GM-CSF, RANTES, MCP-4, CTACK/CCL27, IFN-g, INFa, CD23, CD-40,Eotaxin-2, and TARC.

The analytes can be analyzed in many ways, which can be readily selectedby the ordinarily skilled artisan in accordance with the analyte to beevaluated. A reservoir or collecting container can be applied to thesite for collection of sample, which is then measured using analyticaltechniques. Application of energy can be optimized to maximize analyterecovery. It may be desirable for certain applications to maintain therelative levels of the analyte to other components of the sample.Exemplary assay methods include but are not limited to gelelectrophoresis, agar plating, enzymatic testing, antibody-based tests(e.g., western blot tests, Enzyme-Linked Immuno Sorbent Assay (ELISA),lateral flow assays, and the like), thin layer chromatography, HPLC,mass spectrometry, radiation-based tests, DNA/RNA electrophoresis,(UV-VIS) spectrophotometry, flow assays, and the like.

A quantitative measurement of the presence of tissue constituents in theliquefied. tissue sample can assess the extent of tissue liquefaction.Such an internal calibration can be accomplished by measuring one ormore optical properties of the liquefied tissue sample such asabsorbance, transmittance, scattering, or fluorescence emission uponbeing irradiated by a source emitting electromagnetic waves. Additionalsample parameters such as gravimetric-weight, total protein content, pH,and electrical conductance can be used for calibrating the extent ofliquefaction. Further, measurement of tissue properties such asthickness, rate of water loss, and electrical conductivity can be used.Direct measurement of the concentration of one or more sampled analytessuch as β-actin, β-tubulin, GAPDH (glyceraldehyde 3-phosphatedehydrogenase), LDH (lactate dehydrogenase or any otherabundantly-present biotnolecule whose concentration is expected toremain constant in the tissue, can be used for calibrating the extent oftissue liquefaction. Analytes could also be quantified usingimmunological based assay (i.e. radioimmuno; Eliza; FACs).

Tissue Cells And Microorganisms

In addition to the analytes described above, whole cells of tissue underanalysis, as well as a variety of microorganisms, can be detected intissues of interest using the devices, methods and systems disclosedherein. Tissue cells and most microorganisms are much larger than theanalytes described above, and their extraction from a tissue of interestcan be accomplished using various embodiments of the current invention.Pathogenic and nonpathogenic bacteria, virus, protozoa, and fungi playwell-known roles in various infectious diseases, and their detection canfacilitate a diagnosis of a disease caused by the microorganism (e.g.,tuberculosis, herpes, malaria, ringworm, etc.). The disease stateexhibits either the presence of a novel microorganisms or an alterationin the proportion of resident microorganisms. When a subject issuspected of having an infection with such a microorganism, the devices,methods and systems disclosed herein can be used to quantify or detectthe presence or absence of a microorganism, and facilitate diagnosis ofthe condition.

Non-pathogenic microorganisms are normally present in healthy tissues(“normal flora”), and can play a role in many bodily functions andmaintenance of health of a subject. Detection of these normal floramicroorganisms (e.g., bacteria) in a tissue of interest can also beaccomplished with the current method and device. A subject's tissue canbe sampled and analyzed using the devices, methods and systems disclosedherein to examine the various microorganisms that are naturally present.When a subject is suspected to have an abnormal condition, tissue of thesubject can be sampled according to the devices, methods and systemsdisclosed herein to detect the presence or absence of a change in aprofile of non-pathogenic microorganisms relative to that of a normal,healthy subject. A change in this microorganism profile can facilitatediagnosis of a condition of interest in the subject.

In some embodiments, tissues can be liquefied to recover their cells ormicroorganisms residing therein. Application of the present devices,methods and systems using energy provide for collection of bacteria fromskin of a subject into a collection medium which may optionally containan LPA. For example, application of ultrasound energy to the tissue ofinterest using tris-HCl or PBS is sufficient to collect bacterialmicroflora. In general, use of a device utilizing this method involvesapplication of a sufficient level of ultrasound energy so as to dislodgemicroorganisms from the tissue and enter the collection medium, which isthen collected for subsequent analysis, which may include culturing themedium to determine whether certain microorganisms are present, directlyassaying the medium (e.g., using ELISA techniques, e.g., involvingmicroorganism-specific antibodies, e.g. involving a latex agglutinationtest, e.g., using a nucleic-acid-based diagnostic assay including thepolymerase chain reaction hybridization, DNA sequencing method), or acombination of these approaches. Detection of microorganisms in themedium facilitates diagnosis of a condition of interest. Furthermore, ahigh yield collection of microorganisms could shorten or eliminate aprocess to amplify the number of nucleic acids for diagnostics.

The invention described herein can also be used to collect cells fromthe tissue. Application of energy with an appropriate LPM that liquefiestissues without disrupting cell membranes can be used to harvest wholecells, including viable whole cells from tissues. LPM in this case maycomprise chemicals including but not limited to ion chelating agentssuch as EDTA or enzymes such as trypsin to dislodge the cells,Similarly, with changes in parameters of energy and/or LPM as discussabove, the devices, methods and systems of the present disclosure can beused to collect nuclei or other cellular organelles.

Tissue Of Interest

A variety of tissues are well suited to the devices, methods and systemsdisclosed herein. These tissues include but are not limited to skin,mucosal membranes (nasal, gut, colon, buccal, vagina etc.) or mucus,breast, prostate, eye, intestine, bladder, stomach, esophagus, nail,testicles, hair, lung, brain, pancreas, liver, heart, bone, or aortawall. In one embodiment, the tissue is skin, which can be skin of theface, arms, hands, legs, back, or any other location. While skin andmucosal surfaces are highly accessible for performing liquefaction,liquefaction devices, methods and systems described in this disclosurecan be designed to readily adapt to various internal tissues listedabove. Exemplary devices specific to internal tissues that can find usein the methods disclosed herein include those disclosed in U.S. Pat. No.5,704,361, U.S. Pat. No. 5,713,363, and U.S. Pat. No. 5,895,397, each ofwhich are incorporated herein by reference in their entirety.

In some embodiments, the tissue of interest is other than a tumor or atissue suspected of being a tumor. Where the devices, methods andsystems disclosed herein are applied to detection of a microorganism,the tissue of interest is one suspected of containing a microorganism(e.g., a tissue suspected of having an infection, particularly a deeptissue infection, e.g., infection of the dermal and/or subdermal layersof the skin, including such layers of mucosal membranes).

Method Of Use

The methods disclosed herein can be used for a broad range of tissueevaluations, including assessment of the presence or absence of ananalyte(s) of interest to facilitate diagnosis of a condition ofinterest. In some embodiments, the methods find use where, for example,the patient presents with clinical signs and symptoms suggestive of oneor more conditions, where the methods disclosed herein can facilitate adifferential diagnosis.

In certain embodiments, the current invention provides methods thatinvolve comparing a test analyte profile generated from a patient sampleto a reference analyte profile. A “reference analyte profile” or“analyte profile for a reference tissue” generally refers to qualitativeor quantitative levels of a selected analyte or set of 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more analytes, which are characteristic of a condition ofinterest. Exemplary conditions of interest for which a reference analyteprofile may be provided include, but are not limited to, normalreference analyte profile (e.g., healthy tissue (i.e., absence ofdisease), general tissue health, acceptable or tolerated levels of ananalyte a drug, environmental contaminant, etc.), disease referenceanalyte profile (e.g., an analyte profile characteristic of the presenceof for example, microbial infection (e.g., bacterial, viral, fungal, orother microbial infection), localized diseases in tissues (e.g.,dermatitis, psoriasis, cancers (prostate, breast, lung, etc.),urticaria, etc.), systemic diseases manifested in tissues (e.g.,allergies, diabetes, Alzheimer's disease, cardio- vascular diseases, andthe like); etc.), environmental contaminant reference analyte profile(e.g., an analyte profile characteristic of the presence of unacceptablyhigh levels of an environmental contaminant (e.g., warfare agent,pollens, particulates, pesticides, etc.), drug reference analyte profile(e.g., an analyte profile characteristic of therapeutic levels of adrug, drug-of-abuse (e.g., to facilitate assessment of drug-of-abuse),etc.); and the like. Reference analyte profiles may include analytesthat are members of one or more classes of analytes (e.g., proteins(e.g., antibodies, cancer biomarkers, cytokines,cytoskeletal/cytoplasmic/extra-cellular proteins, and the like), nucleicacids (DNA, RNA), lipids (which include ceramides, cholesterol,phospholipids, etc.), biologically-derived small molecules, drugs (e.g.,therapeutic drugs, drugs- of-abuse), environmental contaminants, warfareagents, etc.) or members of a subclass of analytes (e.g., antibodies,phospholipids). Reference analyte profiles of a given condition ofinterest may be previously known in the art or may be derived from thetissue using the methods described in this invention. Reference analyteprofiles can be stored in electronic form (e.g., in a database) toprovide for ready comparison to a test analyte profile to facilitateanalysis and diagnosis.

A “test analyte profile” or “analyte profile for a tissue of interest”refers to qualitative or quantitative levels of a selected analyte orset of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more analytes, to facilitatediagnosis or prognosis of a condition of interest. A test analyteprofile may include analytes that are members of one or more classes ofanalytes (e.g., proteins, nucleic acids, lipids, biologically-derivedsmall molecules, drugs (e.g., proteins (e.g., antibodies, cancerbiomarkers, cytokines, cytoskeletal/cytoplasmic/extra-cellular proteins,and the like), nucleic acids (DNA, RNA), lipids (which includeceramides, cholesterol, phospholipids, etc.), biologically-derived smallmolecules, drugs (e.g., therapeutic drugs, drugs of abuse),environmental contaminants, warfare agents, etc. or members of asubclass of analytes (e.g., antibodies, phospholipids). In general, theanalytes selected for analysis to generate a test analyte profile areselected according to analytes of a desired reference analyte profile.Comparison of a test analyte profile to an appropriate reference analyteprofile facilitates determining the presence or absence of the conditionor state of interest, e.g., by assessing whether there is a substantial“match” between a test analyte profile and a reference analyte profile.

Methods for generating reference and test analyte profiles of a selectedanalyte or set of analytes can be accomplished using methods availablein the art, and will be selected according to the analyte(s) to beassessed.

The current methods can be used for a broad range of tissue evaluations.Energy-assisted tissue liquefaction can provide a quantitativeevaluation and profile of normal tissue. Comparison of the normal tissueprofile with a profile of tissue under investigation can facilitatediagnosis of changes in tissue microenvironment up/down-regulation ofseveral proteins, lipids, nucleic acids, small molecules, drugs, etc.)which can indicate various diseased conditions such as allergies,cardio-vascular disease, dermatitis, etc. The methods can also be usedas a tool for monitoring tissue recovery and evaluating therapeuticefficacy of various treatments (as in monitoring of therapy, which canbe combined with modification of therapy as desired or needed). Theanalyte profiling methods can also provide tools for the personal-careindustry for evaluation of topical formulations (e.g., as in cosmetics).This methodology can be utilized for determining pharmacologicparameters by liquefying tissues and detecting the drug moleculestherein. in a similar manner, rapid and routine testing of chemicals,bio-hazardous contaminants, and drugs-of-abuse can also bequantitatively accomplished. The methods can also be used for sensitivedetection and diagnosis of pathogenic microflora.

In certain embodiments, the current methods provide a profile of normaltissue, wherein normal tissue is defined by the absence of the abnormaltissue condition of interest. Energy is applied to the normal tissue,e.g., by ultrasound exposure or abrasion, in the presence of aliquefaction-promoting agent. Various tests are performed upon theliquefied tissue sample to isolate and identity the analytes present inthe tissue.

In certain embodiments, the methods can be applied to facilitatediagnosis of various tissue diseases which are characterized by aquantitative evaluation of a change in the tissue microenvironment. Thisevaluation is performed by comparing an analyte profile of a referencetissue (e.g., a reference analyte profile, which may be stored in adatabase) with the analyte profile of the tissue of interest (i.e., thetest analyte profile). The quantitative presence or absence of a certainanalyte or set of analytes present in a tissue under investigation, whencompared to the quantitative presence or absence of the same analytes ina reference tissue will indicate the presence or absence of a particulardisease, and thus facilitate diagnosis of the condition. The referenceanalyte profile can be one characteristic of tissue which is known tonot be affected with the disease in question, or can be a referenceanalyte profile characteristic of the disease in question for the tissuein question.

In one embodiment, the tissue under investigation is skin and/or mucosalmembranes, and the quantitative test analyte profile is compared to areference analyte profile to determine the presence or absence of adisease such as allergy, urticaria, microbial infection, auto-immunedisease, cardiovascular disease, or cancer.

In certain embodiments, this method can be used to monitor tissuerecovery. This monitoring is performed by comparing an analyte profileof reference tissue with the analyte profile of tissue underinvestigation. The quantitative presence or absence of a certain analyteor composition of analytes present in a tissue under investigation, whencompared to the quantitative presence or absence of the same analytes ina reference tissue can indicate whether or not the tissue is returningto its healthy state. The reference tissue is usually tissue that is ina healthy state.

In certain embodiments, the current methods can be used to evaluate thetherapeutic effect of various treatments, including bioavailability oftherapeutics in tissues of interest. The analyte in the liquefied tissuesample can be quantified to indicate how much of the analyte is presentin the tissue. The quantitative presence or absence of a certain analyteor composition of analytes present in a tissue under investigation, whencompared to the quantitative presence or absence of the same analytes ina reference tissue, can indicate whether or not the dosed therapeuticagent is staying in the specific tissue or body long enough to achieveits desired effect. The reference tissue is usually tissue that is in ahealthy state.

In certain embodiments, the methods disclosed herein can be used toevaluate therapeutic formulations on a tissue such as skin,specifically, whether component(s) of a formulation (e.g., lotions,creams, salves, and the like) are being absorbed by the tissue, and ifthe amount delivered is therapeutically effective. In certainembodiments, the methods disclosed herein can include a closed loopsystem, in which the same system can apply the therapeutic formulation,liquefy the analytes, analyze the analyte profile, and adjust thedelivery of the formulation accordingly. The reference tissue in thiscase ⁻would be healthy tissue, or tissue at various levels of recoveryfrom the condition that the therapeutic formulation was treating.

In certain embodiments, the current methods can be used to determine theanalyte profile for use in determining pharmacological parameters orefficacy of pharmaceutical agents. The presence or absence of certainanalytes (e.g., immune system responders, cytokines) can be used tocorrelate certain dosages of pharmaceutical agents to biologicalparameters, including but not limited to AUC, clearance, and half-life.

In certain embodiments, the methods disclosed herein can be used todetect the presence or absence of certain chemicals, including but notlimited to bio-hazardous contaminants, warfare agents, illicit drugs,known pharmaceutical agents, and the like. Such methods find use in, forexample, law enforcement, regulation of doping in competitive sports,evaluation of exposure and/or risk of disease as a result of exposure totoxins or contaminants, and the like.

In certain embodiments, the current methods can be used for detecting ordiagnosing pathogenic microbes (e.g., bacteria, fungi, viruses, and thelike). Current methodologies for microbial diagnostics in tissues, suchas replica plating, swabbing, and washing, are unattractive due to largevariability and low dispersion of extracts, which leads to decreasedsensitivity and high protocol-dependency. Various tests can be performedupon the liquefied tissue sample to isolate and identify the microbialanalytes present in the tissue. in certain embodiments, these testsinclude plating on agar plates.

Drug Delivery

The present invention provides a method and device involvingliquefaction of a tissue so as to control and enhance the flux of drugsinto or through the tissue. The method includes the steps of 1) applyingenergy and a liquefaction promoting medium to a tissue where transportis desired of a subject; and 2) delivering one or more drugs into orthrough the tissue to be liquefied continuously or repeatedly. Themethod may further include re-liquefy the tissue over the period of timeduring which transport occurs. The method comprising liquefying a tissuecan perturb the barrier properties of a tissue or biological surface,leading to reducing the resistance to the drug's passage. The advantageof the present invention is that the rate and efficiency of transfer isboth improved and controlled. Drugs which would simply not pass throughthe biological surfaces, or pass at a rate which is inadequate orvariable over time, are forced into the biological surfaces when energyin combination of a LPM is applied. By controlling the mode, intensityand time of energy application and formulation of a LPM, the rate oftransfer is controlled.

The transport of drugs can be modulated or enhanced by the simultaneousor subsequent application of a secondary driving force such as chemicalpermeability or transport enhancers, convection, osmotic pressuregradient, concentration gradient, iontophoresis, electroporation,magnetic field, ultrasound, or mechanical pressure.

Enhancement of the disclosed method was demonstrated by the followingnon-limiting example employing ³H-labelled Acyclovir and Inulin. Therequired type, length of time, and intensity of energy and formulationof a LPM are dependent on a number of factors including the type oftissues and the property of drugs, which varies from species to species,with age, injury or disease, and by location on the body.

Drug To Be Administered

Drugs to be administered include a variety of bioactive agents, but arepreferably proteins or peptides. Specific examples include insulin,erythropoietin, and interferon. Other substances, including nucleic acidmolecules such as antisense, siRNA and genes encoding therapeuticproteins, synthetic organic and inorganic molecules includinganti-inflammatories, antivirals, antifungals, antibiotics, localanesthetics, and saccharides, can also be administered. The drug willtypically be administered in an appropriate pharmaceutically acceptablecarrier having an absorption coefficient similar to water, such as anaqueous gel. Alternatively, a patch can be used as a carrier. Drug canbe administered in a gel, ointment, lotion, or suspension.

In one embodiment, the drug is in the form of or encapsulated in adelivery device such as liposome, lipid vesicle, emulsion or polymericnanoparticles, microparticle, microcapsule, or microsphere (referred tocollectively as microparticles unless otherwise stated). These can beformed of polymers such as polyhydroxy acids, polyorthoesters,polyanhydrides, and polyphosphazenes, or natural polymers such ascollagen, polyamino acids, albumin and other proteins, alginate andother polysaccharides, and combinations thereof. The microparticles canbe coated or formed of materials enhancing penetration, such aslipophilic materials or hydrophilic molecules, for example, polyalkyleneoxide polymers, and conjugates, such as polyethylene glycol.

Administration Of Drug

The drugs are preferably administered, using the liquefaction devicesmentioned, to the tissues at a site selected based on convenience to thepatient as well as to achieve desired treatment results. A variety oftissues including biological surfaces are well suited to the currentmethod. These tissues include but are not limited to skin, mucosalmembranes (nasal, gut, colon, buccal, intestine, vagina, etc.). In oneembodiment, the method of the current invention is preferablyadministered to the skin of the face, arms, hands, legs, back, or anyother location. While skin is highly accessible for performingliquefaction, the devices described in this disclosure can be designedto readily adapt to various internal membranes listed above.

In some embodiment, the tissue to be administered is a diseased tissuesuch as infectious organs, tissues that is inflamed, and solid tumors.In a certain embodiment, the present invention comprises using theliquefying devices on the healthy tissues in the vicinity of and/or thediseased tissue, and delivering drugs across the healthy tissues and/orinto the site of the diseases. Steroids such as corticosteroids and manyof chemotherapeutic agents including estramustine phosphate, paclitaxel,and vinblastine have potentially severe side effects. Hence, if givensystemically, they are likely to cause undesirable side effects. Thisproblem is overcome by delivering these drugs locally to the diseasetissues, Other indications include delivery of drugs into abnormal skinsuch as psoriasis, atopic dermatitis, and scars.

In some embodiments, the current invention is used to enhance thepassage of a compound such as a large molecular weight or polar moleculethrough the tissue such as skin, mucosal membranes nasal, gut, colon,intestine, buccal, vagina etc.). Greater control and drug utilizationare achieved by increasing the rate and directional control of theapplied drug. The percentage of drug which quickly enters thebloodstream is increased accordingly and undesirable side effects areavoided. Drugs through the tissues stated above are infused into thebloodstream at an optimal rate.

Liquefaction Promoting Medium (LPM) For Drug Delivery

A LPM is also an important component for drug delivery. The design ofthe LPM for drug delivery is overlapping somewhat to that of the LPM forsample collection. The LPM can be designed to serve one or more of thefollowing five purposes: a) it couples energy to a tissue, b) itfacilitates liquefaction of the tissue, c) it storages drugs to bedelivered into the tissue, d) it increases the solubility of the drugs,and e) it inhibits degradation of the drugs such that their biologicalor chemical activity is retained.

The LPM may also contain a drug prior or during tissue liquefactionprocess. In an alternate embodiment, application of energy and the LPMwhich excludes a drug can be used for liquefying a tissue, andsubsequently a drug in an appropriate carrier such as a patch can beapplied on a site of the tissue to be liquefied.

Kits

The present disclosure also encompasses kits for practicing the currentmethods, The subject kits can include, for example, the entire energyapplication device and a liquefaction- promoting agent to liquefytissues of interest, reagents for conducting assays to detect andanalyze (qualitatively or quantitatively) the presence or absence oftissue analytes in the liquefied tissue sample generated through methodsdisclosed herein. The various components of the kit may be present inseparate containers, or certain compatible components may bepre-combined into a single container, as desired.

In addition to the above-mentioned components, the kits typicallyfurther include instructions for using the components of the kit topractice the methods. The instructions for practicing the subjectmethods are generally recorded on a suitable recording medium. Forexample, the instructions may be printed on a substrate, such as paperor plastic, etc. As such, the instructions may be present in the kits asa package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging orsub-packaging) etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g. CD-ROM, diskette, etc. In yet otherembodiments, the actual instructions are not present in the kit, butmeans for obtaining the instructions from a remote source, e.g. via theinternet, are provided. An example of this embodiment is a kit thatincludes a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions is recorded on a suitablesubstrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep, or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

Example 1

Sampling of Skin By An Abrasive Energy-Based Device

Referring to FIGS. 7A through 7F, an abrasive energy-based tissueliquefaction device for sampling of skin tissue is described. Device isassembled from three components—751 (assembly of device housing 701containing motor 704 and electrical conductivity components 705 and706); 752 (disposable assembly of LPM cartridge 708, collectioncontainer 707 and needle 709); and 753 (disposable assembly of LPMhousing 712, abrasive pad 711 and shaft 710) (FIG. 7A). The assembleddevice is placed against a pre-identified region of interest on skin713, such that abrasive pad 711 is facing skin 713 (FIG. 7B). Slidingplunger 702 located on top of the device is pushed towards skin, whichpushes needle 709 into LPM cartridge 708, breaking its sterile seal andtransfers LPM into housing 712. Sliding plunger 702 also energizes motor704 through battery pack 703, setting the shaft 710 and abrasive pad 711in rotary motion against skin tissue 713 (FIG. 7C). As skin tissue isliquefied, tissue components are dissolved in LPM contained in housing712. Electrical conductivity of skin tissue 713 is also simultaneouslymeasured using sliding contact 705 fastened to shaft 710 as measurementelectrode and reference electrode 706. Once the safe energy exposurelimit is reached as determined by threshold electrical conductivity,motor 704 stops. Sliding plunger 702 is further pushed towards skin suchthat needle 709 punctures a pre-vacuumized sample container 707, whichaspirates the sample from housing 712 in it (FIG. 7D). The device isremoved from skin and disassembled (FIG. 7E). The device component 752is further dissembled and sample container 707 is processed fordetection of analytes (FIG. 7F).

Example 2

Sampling of Skin by a Microneedle-Based Device

Referring to FIGS. 8A through 8D, a microneedle-based tissueliquefaction device for sampling of skin tissue is described. The deviceis placed against a pre-identified region of interest on skin 807, suchthat microneedle bearing patch 805 is facing skin 807 (FIG. 8A). Slidingplunger 801 located on top of the device is pushed towards skin 807 suchthat LPM soaked sponge 804 is squeezed and releases LPM into housing 803(FIG. 8B). Consequently, microneedles in patch 805 and housing 803 atthe skin interface are filled with LPM. To initiate liquefactionprocess, sliding plunger 801 is further pushed into skin tissue 807leading to insertion of microneedles 805 into skin tissue 807 (FIG. 8C).As skin tissue is liquefied, tissue components are dissolved in LPMcontained in housing 803. Upon completion of skin liquefaction,pre-vacuumized sample container 802 is pushed towards skin tissue 807such that needle 806 punctures sample container 802 resulting inaspiration of sample from housing 803 in it (FIG. 8D). The device isremoved from skin and disassembled. Sample container 802 is retrievedfor analyte analysis and the rest of the device components are disposedof.

Example 3

Reservoir Housing For Capturing Tissue Analytes

Referring to FIGS. 9A, 9B, 9C and 9D a design for a reservoir housing tocapture tissue analytes from liquefied tissue samples is described. Thereservoir housing 901 is intended to be used with energy-applicationdevices described herein, as a container to collect the liquefied tissuesample (FIG. 9A). The housing is coated with capture substrates 902which selectively bind to tissue analytes 903 present in the sample(FIG. 9B). Upon sufficient incubation of the tissue sample, the sampleis discarded while the analytes 903 are held in the housing (FIG. 9C).The analytes are eluted by an elution buffer in the housing forsubsequent capture of the analytes as a separate sample 904 (FIG. 9D).Alternatively, the housing can be integrated in an analytical tool foranalyzing the bound analytes 903.

Example 4

Surfactant Formulations For Enhanced Tissue Solubilization and ProteinFunctionality Retention

Unique surfactant formulations were identified that make up theliquefaction promoting medium (LPM) according to the definitiondisclosed in this text. A library of 153 binary surfactant formulationswas created using 19 surfactants belonging to four distinct categories:(i) anionic surfactants (sodium lauryl sulfate (SLS), sodium laurethsulfate (SLA), sodium tridecyl phosphate (TDP), sodium deoxycholate(SDC), sodium decanoyl sarcosinate (NDS), sodium lauroyl sarcosinate(NLS), sodium palmitoyl sarcosinate (NPS)); (ii) cationic surfactants(octyl trimethyl ammonium chloride (OTAB), dodecyl trimethyl ammoniumchloride (DDTAB), tetradecyl trimethyl ammonium chloride (TTAB)); (iii)zwitterionic surfactants (3-[(3-cholamidopropyl) dimethylammoniol]1-propane sulfonate (CHAPS), 3-(decyl dimethyl ammonio) propanesulfonate (DPS), 3-(dodecyl dimethyl ammonio) propane sulfonate (DIPS));(iv) nonionic surfactants (polyethylene glycol dodecyl ether (830),polyoxyethylene 23-lauryl ether (835), polyoxyethylene 10-cetyl ether(856), polyoxyethylene 2-stearyl ether (872), polyethylene glycol oleylether (893), nonylphenol polyethylene glycol ether (NP9)). Only ahandful of surfactants from these categories (for example, nonionicsurfactants) have been traditionally utilized for extracting functionaltissue proteome. Additionally, these surfactants are highly limited intheir ability to efficiently solubilize tissue constituents. As such,across all surfactant types, extraction potential and bioactivitypreservation of tissue constituents are largely considered as mutuallyconflicting properties. By combining nonionic surfactants with othertypes of surfactants that have been previously described for their highsolubilization ability (anionic, cationic and zwitterionic surfactants),we show the discovery of new families of surfactant formulations thatsimultaneously possess superior solubilization as well as non-denaturingcapabilities.

The surfactant library was first screened for identifying non-denaturingsurfactant formulations that retain protein bioactivity in extracts, andsubsequently ranked for the ability of formulations to solubilize tissueproteins. FIG. 10A shows the potency of 153 surfactant formulations topreserve the specific functionality of a model protein IgE antibody.Specifically, binding ability of IgE antibody with ovalbumin was tested.The x-axis in this figure represents the formulation index unique toeach binary formulation. The y-axis represents % IgE bioactivityretention, defined as the fractional IgE binding activity in surfactantformulation compared with IgE binding activity when surfactants in puresolvent (phosphate buffered saline, PBS). The formulations spanned awide range of denaturing potentials. Surprisingly, an increasing numberof denaturing surfactants upon combination with gentler nonionicsurfactant yielded a high synergistic gain in IgE functionalityretention. Non-denaturing potential, averaged over all binary surfactantformulations was found to be significantly higher than their constituentsingle surfactant formulations (p <0.006; two-tailed heteroscedasticstudent's t-test); further demonstrating unique synergic interactions.

Surfactant formulations exhibiting high bioactivity retention 90%) werefurther screened for their ability to extract tissue proteins inconjunction with a brief sonication treatment. Porcine skin was used asa model tissue for these studies. While a majority of formulationsrevealed an extraction potential close to 0.1 mg protein per cm² of skintissue, only a couple of formulations achieved protein extractionexceeding 0.3 mg/cm² (FIG. 10B).

The leading candidates screened from the surfactant library generallyresulted in formulations that were exceptionally non-denaturing, yetmore effective in solubilizing tissues than some of the most widely usedextraction surfactants reported in the literature. FIG. 10C compares theleading surfactant formulation- 0.5% (w/v) DPS-B30, with 1% (w/v) SDSfor skin sampling. Despite a moderate extraction ability (0.16 ±0.07mg/cm²), SDS is highly denaturing which results in a low yield offunctional protein recovery (product of fractional bioactivity retainedand total extracted protein). In contrast, 0.5% (w/v) DPS-1330formulation not only extracts more skin proteins (0.48 ±0.12 mg/cm²) butalso preserves protein activity, amounting to an excess of 100-foldenhancement in expected functional protein recovery over SDS. Similarly,more than 10-fold of protein recovery were accomplished over commonlyused non-denaturing surfactant 1% (w/v) Triton X-100 and PBS.

Example 5

Bioactivity Retention Under Stress

We show that unique surfactant formulations, or LPMs (as identified bymethod described in Example 1) additionally protect a variety ofanalytes under stress. We specifically show denaturing effects ofmechanical energy such as ultrasound exposure (a commonly knowndenaturant to biomolecules), can be neutralized with the use of uniquesurfactant formulations.

In separate experiments, a globular protein (IgE) and two representativeenzymes—lactate dehydrogenase (LDH) and beta-galactosidase (β-Gal) weredissolved in 0.5% (w/v) DPS-B30 surfactant formulation and sonicated todetermine retention of protein bioactivity over time. Proteins dissolvedin saline (PBS) were prepared as comparative controls. A progressivelysharp decrease in functionality was observed for IgE dissolved in PBS;however, 0.5% (w/v) DPS-B30 formulation, surprisingly, extendedprotection to IgE proteins towards ultrasonic denaturing stress (FIG.11A). Irrespective of ultrasound treatment, IgE dissolved in SDS showedcomplete state of denaturation. Similar trends were observed on extendedpreservation of enzymatic activities for LDH and β-Gal prepared in 0.5%(w/v) DPS-B30 formulation (FIG. 11B). Protein preparation in PBSresulted in significant loss of bioactivity (p <0.006; two-tailedheteroscedastic student's t-test), amounting to a fractional bioactivityof 16.7% (IgE), 70.8% (LDH) and 68.7% (β-Gal) after 3 minutes ofsonication.

Example 6

Tissue Sampling and Molecular Diagnostics

The ability of ultrasonic exposure in the presence of LPM (salinesolution of 0.5% (w/v) DPS-B30) to sample a variety of functionaldisease biomarkers from tissues was demonstrated.

Sampling of allergy-specific IgE antibodies from the skin of miceallergic to egg was demonstrated. Six to eight weeks old female BALB/CJmice were purchased from Charles River Labs (Wilmington, Mass.) andmaintained under pathogen-free conditions. Allergic reaction was inducedin mice by an epicutaneous exposure protocol. After anesthesia with1.25-4% isofluorane in oxygen, the skin on the back of the mice wasshaved and then tape stripped 10 times (Scotch Magic tape, 3M HealthCare, St Paul, Minn.) to introduce a standardized skin injury. A gauzepatch (1 cm×1 cm) soaked with 100 μL of 0.1% OVA was placed on the backskin and secured with a breathable elastic cloth-based adhesive tape.The patches were kept affixed for 1 week. The whole experiment compriseda total of three 1-week exposures with a 2-week interval between eachexposure week. Sampling was performed by gluing a custom made flangedchamber (skin exposure area of 1.33 cm²) to the shaven skin area with aminimal amount of cyanoacrylate-based adhesive. The chamber was filledwith 1.8 ml of 0.5% (w/v) DPS-B30 surfactant formulation and 20 kHzultrasound was applied at 50% duty cycle, 2.4 W/cm² for 5 minutes. Skinbiopsies of ultrasound treated or untreated eczema skin sites wereobtained, and skin homogenate samples were prepared as positivecontrols. FIG. 12A shows that ultrasound-assisted sampling successfullysampled significantly more amount of allergy-specific IgE antibodiesfrom allergic mice skin as compared to healthy mice. Expectedly, nodifference was seen in the amount of IgG antibodies in the samples fromallergic and healthy mice skins.

Sampling of cholesterol from mouse skin was also demonstrated. Withsimilar procedures as described in above paragraph, skin samples withthe ultrasound procedure were collected. Skin homogenates were preparedas positive controls from biopsies collected from untreated skin. Skincholesterol is an important biomarker for diagnosing cardiovasculardisease [1]. FIG. 12B shows that ultrasound-assisted samplingsuccessfully samples cholesterol from skin and the amount sampled iscomparable to the cholesterol present in skin homogenate.

Lastly, sampling of bacterial genome from porcine skin was demonstrated.Tissues, particularly skin and mucosal membranes are colonized by adiverse set of microorganisms including bacteria, fungi and viruses[2-5]. Accurate diagnosis of bacterial infection leads to appropriatepatient management, providing information on prognosis and allowing theuse of a narrow-spectrum antibiotics [6-8]. Thus, definitivemicroorganism detection is essential for diagnosis for treatment ofinfection and trace-back of disease outbreaks associated with microbialinfections. Accurately obtaining samples which represent microorganismson skin, however, is a major challenge [2]. The most practical method ofcollection would be swabbing because it is simple, quick and noninvasive[3, 9]. However, swabbing has several limitations including poorrecoveries of the microorganisms and lack of a standardized protocol,which suggests it either does not accurately represent themicroorganisms on the skin or provide quantitative data.Ultrasound-assisted sampling can effectively address these limitations.In particular, excised porcine skin was sampled by swabbing with acotton ball soaked in saline (PBS), and by ultrasound-assisted samplingwith 0.5% (w/v) DPM-Brij30 as LPM in separate experiments. The bacterialgenome was purified from each sample by standard phenol-chloroformextraction method. Briefly, samples were first incubated in a solutionconsisting of 20 mM tris-HCl at pH 8.0 (BP154-1, Fisher Scientific), 2mM EDTA(BP120-500, Fisher Scientific), 1.2% Triton X-100 (BP151-100,Fisher Scientific), and 20 mg/ml lysozyme (62970-IG-F, Sigma-Aldrich)for 30 min at 37° C. [9]. Subsequently, samples were incubated for 3hours at 37° C. in a solution consisting of 0.1 mg/ml Proteinase K(P2308-25MG, Sigma-Aldrich), 0.5% (w/v) sodium lauryl sulfate (S529,Fisher Scientific), and 100 mM sodium chloride (BP358-1, FisherScientific). Genomic DNA was then extracted with an equal volume ofphenol (P4557, Sigma-Aldrich), followed by extraction withphenol/chloroform/isoamyl alcohol, 25:24:1 (P2069, Sigma-Aldrich). TheDNA was precipitated by incubation with ethanol and centrifugation for20 min. The DNA pellets were washed twice with 70% ethanol, allowed todry, and re-suspended in 80 μl of tris buffer. The amount of bacteriasampled by each methodology was evaluated by determining the presence ofthe conserved 16S bacterial gene in each sample usingquantitative-polymerase-chain-reaction (qPCR). FIG. 12C shows thatultrasound-assisted sampling sampled at least 7-fold higher amount ofbacterial genome from skin than the conventional cotton swabbingprocedure.

Example 7

Buffer Design of LPMs Compatible With Nucleic-Acid-Based Tests

To ensure compatibility of the liquefied tissue samples with subsequentanalysis, the components of LPMs have to be carefully chosen.Compatibility of several LPM components with nucleic-acid-basedanalytical technique was tested. Specifically, the compatibility of LPMcomponents with qPCR the most common gene-based test was evaluated bymeasuring the test's ability to amplify plasmid DNA added in differentLPMs.

Ten million copies of Luciferase plasmid (E1741, Promega Corp.) werespiked in 10 μl of different solutions: (i) water, (ii) 0.91% (w/v)sodium chloride (BP358-1, Fisher Scientific) in water, (iii) PBS (P4417,Sigma-Aldrich), (iv) 10 mM tris-HCl, pH 7,9 (BP154-1, FisherScientific), (v) 0.075 M sodium phosphate buffer, pH 7.9, derived fromsodium phosphate monobasic monohydrate and sodium phosphate dibasic(S9638-25G, 57907-100G, Sigma- Aldrich), and (vi) 0.5 mM EDTA(BP120-500, Fisher Scientific) in water. The solutions were combinedwith 10 μl of PCR reaction buffer. Luciferase amplification primers were5′-GCC TGA AGT CTC TGA TTA AGT-3′ (SEQ. ID NO. 1) for the forward primerand 5′-ACA CCT GCG TCG AAG-T-3′ (SEQ. ID NO. 2), for the reverse primer,creating an amplicon of 96 by [10]. Amplification reactions wereperformed in a 20 μl solution containing MgCl₂ at 1.5 mM, primers at 0.2μM (each), and 0.2 mM dNTPs in PCR buffer and 0.025 units/μl of Taqpolymerase (1-034, Invitrogen) and SYBR-green (S-7563, Invitrogen) at1:45,000. Aliquots of plasmid DNA were diluted in water to generate astandard curve. Analysis was performed on iCycler PCR machine (Bio-RadLaboratories, Inc.) using optical grade 96-well plates. Thermal cycle ofthe reaction was set as follows: initial denaturation at 95° C. for 3min, followed by 40 cycles of denaturation at 95° C. for 30 sec, 30 secannealing at 60° C., and 30 sec elongation at 72° C., all followed by afinal extension of 10 min at 72° C. For each sample, three replicateswere performed. For each buffer, the compatibility was calculated bycomparing with the control (plasmid DNA in water).

FIG. 13 shows that sodium chloride. PBS, and sodium phosphate buffer wasincompatible as detection buffer for quantitative PCR assay as comparedwith control. However, use of tris-HCl or EDTA as butler increased theanalytical assay's detection ability.

Example 8

Compatibility of LPMs With Nucleic-Acid-Based Tests

Compatibility of various LPMs (disclosed in EXAMPLE 1) with existingnucleic-acid-based tests was tested. Specifically, plasmid DNA was mixedwith different LPMs and the ability of qPCR to amplify DNA was assessed.LPMs were prepared by adding surfactants at various concentrations in 10mM tris-HCl buffer. To mimic the process of tissue liquefaction asdisclosed in this text, each LPM was mixed with 0.2 mg/ml of pig skinhomogenate and ten million copies of Luciferase plasmid (E1741, PromegaCorp.) were spiked per 10 μl of LPM. This solution was combined with 10μl of PCR reaction buffer. qPCR was performed according to the protocoldescribed in EXAMPLE 4. Purified plasmid DNA were diluted in atris-1-HCl solution to generate a standard curve. Compatibility of eachLPM was calculated by determining the amount of plasmid amplified byqPCR and comparing it with the control buffer (plasmid DNA in tris-HClwithout surfactant).

FIG. 14 shows that Triton X-100, Brij 30, DMSO, OTAB, OTAB-Brij 30, andDPS-Brij 30 were highly compatible with quantitative PCR; however LPMsconsisting of NLS or NLS-Brij30 failed to amplify the DNA. Notably,DPS-Brij 30 as a LPM effectively samples biomolecules from tissues,retains protein activity and is compatible with analytical methodsincluding ELISA, chromatography and qPCR. Therefore, DPS-Brij 30 is mostdesirable as liquefaction promoting media for analyzing proteins, lipidsand nucleic acids. Triton X-100 and DMSO, which have been known as afacilitator of PCR [11], were consistently shown to effectively producepolymerase-chain reactions; however, they do not yield satisfactorytissue extraction.

Example 9

Identification of Ultrasonic Parameters For Sampling Viable andGenetically-Intact Microorganisms From Tissues

This example describes a nonlethal condition of ultrasound toefficiently collect living microorganisms from tissues. Microorganismscan be collected from tissue by applying various form of energy totissues; however use of high energies is highly detrimental to theviability of microorganisms. Therefore, it is essential to find outnonlethal conditions of energy application for sampling livingmicroorganisms. We describe ultrasound exposure conditions for samplingviable and genetically-intact bacteria from skin.

Bacterial culture of E. Coli strain DH10α (18290-015, Invitrogen) weregrown in Luria-Bertani (BP1426, Fisher Scientific) at 37° C., 250 rpm oras solid culture on Agar plates (37° C.). Culture was harvested bycentrifugation and the resulting pellet was suspended in LPM comprisingof 10 mM tris-HCl, pH 7.9 at a concentration of 10⁹ cells/mi. E. Colicells were quantified with a spectrophotometer (Biophotometer,Eppendorf), and a bacterial culture of 0.25×10⁹ cells/nil was consideredto correspond to an optical density absorbance value of 0.25 at awavelength of 600 nm. One ml of the re-suspended cells was placed in asterilized cylindrical container (internal diameter 20 mm, flat base,1.3 mm wall thickness, 31 mm height). All experiments were performedwith a 600-Watt sonicator (Sonics & Materials, Newtown, Conn.) operatingat a frequency of 20 kHz at 50% duty cycle. The power setting and thetime of ultrasound exposure were varied in this experiment. Thetransducer was lowered into the container until the probe was immersedin the fluid at a distance of 5 mm from the bottom. The transducer wassterilized by 70% ethanol between sonication procedures on differentsamples. After sonication, 10-fold serial dilutions of each sample wereprepared in 10 mM tris-HCl (pH 7.9). 100 μl of sample from each dilutionstep was plated onto Luria-Bertani agar and spread with a sterilespreader. The plates were incubated at 37° C. for 24 h and viablebacterial colony counts were made on the surface of agar plates. Resultswere expressed as percentage reduction in viability relative tonon-sonicated controls. To evaluate integrity of bacterial genome insamples exposed to ultrasound, electrophoresis was carried out. Allsamples were incubated at 56° C. in Proteinase K (19131, Qiagen) and0.5% (w/v) sodium lauryl sulfate (S529, Fisher Scientific), After 1-hincubation, total genomic DNA was extracted by using the DNeasy DNAExtraction Kit (69504, Qiagen). The standard protocol for the kit wasfollowed for all subsequent steps. The purified genomic DNA wasresuspended in 400 μl of Buffer AE and stored at −20° C. until analysis.The purified DNA was electrophoresed for 90 min at 100 V in a 2% (w/v)tris-acetate-EDTA-agarose gel. The gels were stained with SYBR Gold(S11494, Invitrogen) and visualized under UV light.

FIG. 15 shows that viability of E. Coli exposed to ultrasound at anintensity of 1.7 W/cm² for up to 2 min was statistically insignificantto the viability of non-treated E. coli samples. This suggests thatthese ultrasonic liquefaction conditions can be used for samplingbacteria without a major loss of viability. However, samples sonicatedat higher power output exhibited a more rapid decrease with applicationtime, and the cell viabilities were significantly different comparedwith non-treated cells. Even after 1 minute exposure at a higherintensity, viability was reduced to 3.6% (p <0.05). This observation isin agreement with bacterial genome integrity as assessed byelectrophoresis (FIG. 16). No damage to bacterial genome was observedupon sonication for 2 minutes at 1.7 W/cm² (conditions shown to maintaincell viability); however, in contrast, the genomic DNA of E. coli cellssonicated at intensities of 1.7 W/cm² (32% viability) and 2.4 W/cm² (8%viability) for 3 min were highly fragmented as can be seen by theirmigration to lower molecular weight part of the gel. These resultssuggest that collection of living bacteria should be performed at anultrasound intensity of 1.7 W/cm² for up to 2 min.

Example 10

Detection of Living Microorganisms From Tissues

A brief exposure of ultrasonic energy coupled with LPM (tris-HCl buffer)can sample viable bacteria from skin. Skin bacteria sampled byultrasound were quantified by the conventional colony counting assay aswell as real-time quantitative PCR, and evaluated by comparing withstandard sampling methods such as swabbing and the surfactant scrubbingtechnique.

In vitro experiments were performed on porcine skin to assess samplingof skin-resident bacteria. Pre-cut frozen full-thickness porcine skinharvested from the lateral abdominal region of Yorkshire pigs wasprocured in 10 cm×25 cm strips from Lampire Biological LaboratoriesInc., PA. The skin was stored at −70° C., until the experiment. Skinpieces with no visible imperfections such as scratches and abrasionswere thawed at room temperature and cut into small pieces (2.5 cm×2.5cm) and mounted on a Franz diffusion cell (Permegear, Hellertown, Pa.,USA). The receiver chamber of the diffusion cells was filled withphosphate buffered saline (PBS) (P4417, Sigma-Aldrich, St. Louis, Mo.)and the donor chamber (skin exposure area of 1.77 cm²) was filled with 1ml of 10 mM tris-HCl buffer (pH 7.9), which also acted as the couplingfluid between the ultrasound transducer and skin. The ultrasoundtransducer was placed at a distance of 5 mm from the skin surface and anultrasonic intensity of 1.7 W/cm² was applied for 2 minutes. The probewas disinfected with 70% ethanol between experiments on differentsamples. As comparative controls, samples were obtained by swabbing theskin. Cotton swabs (B432, BD Diagnostics) were soaked in sterilizedphosphate-buffered saline before use. The area of the sample site wasstandardized by holding a sterilized metal ring enclosing an area of 3.3cm² onto the skin surface. The skin surface was rubbed gently andrepeatedly for approximately 20 seconds. Each swab was extracted with 1ml of PBS. Skin bacteria were also sampled by the surfactant scrubtechnique of Williamson and Kligman [2, 12]. A sterile metal ring wasfirmly held against the skin surface and 1 ml of 0.1% Triton X-100 in0.075 M phosphate buffer, pH 7.9, was pipette into it. The skin surfacewithin the ring was rubbed firmly for 1 min with a Teflon cell scraperand the resulting sample was collected in a sterile centrifuge-tube. Theprocedure was repeated at the same skin site for two additional timesand samples were pooled together. Serial 10-fold dilutions of eachsample were prepared and 100 μl aliquots from each diluted sample wereplaced on Tryptic Soy agar plates (9, BD Diagnostics) [12]. The plateswere subsequently incubated under aerobic conditions at 37° C. for 24hours and colonies were counted to obtain an estimate of extractionefficiency by calculating the colony-forming unit per unit area ofsampled skin (CFU/cm²). To quantify total bacteria, real-timequantitative PCR was performed based on an amplicon of the 16S rRNAgene. All biological specimens were first incubated in a preparation ofenzymatic lysis buffer (20 mM Tris at pH 8.0, 2 mM. EDTA, 1.2% TritonX-100) and lysozyme (20 mg/mL) for 30 min at 37° C. [9]. Subsequently,samples were incubated for 1 hour at 56° C. in Buffer AL and ProteinaseK from the DNeasy DNA Extraction Kit (Qiagen). The standard protocol forthe kit was followed for all subsequent steps. The DNA eluted by BufferAE was precipitated by incubation with equal volumes of absoluteisopropanol and then centrifuging for 20 min, The DNA pellets werewashed once with 70% ethanol, allowed to dry, and re-suspended in 80 82l of Buffer AE. Negative controls were also prepared using untreatedsterile cotton swabs in PBS. Analysis of the 16S genes was performed onthe iCycler PCR machine (Bio-Rad Laboratories, Inc.) using optical grade96-well plates. A portion of the bacterial 16S gene was amplified usingforward primer 63F (5′-GCA GGC CTA ACA CAT GCA AGT C-3′, SEQ. ID NO. 3)and reverse primer 355R (5′-CTG CTG CCT CCC GTA GGA GT-3′, SEQ. ID NO.4) [9, 13]. A standard curve was constructed by amplifying serialdilutions of genomic DNA from known quantities of E. Coli cells in 10 μlof Buffer AE. 10 μl of purified DNA was mixed with 2 pmol of each primerand Platinum PCR Supermix (11784, Invitrogen) to a final reaction volumeof 20 μl. Thermal cycling was set as follows: initial denaturation at94° C. for 5 min, followed by 32 cycles of a 30 sec 94° C. denaturation,30 sec annealing at 66° C., and 30 sec elongation at 72° C., allfollowed by a final extension of 10 min at 72° C. For each sample, threereplicates were performed.

FIGS. 17A and 17B show comparison of the sampling efficacies ofdifferent techniques. FIG. 17A shows that ultrasonic sampling recoveredapproximately 17-fold higher number of bacteria from skin than cottonswabbing (p <0.05). Notably, counts of the total number of bacteriacollected by ultrasound did not differ significantly from the positivecontrol (surfactant scraping method). The effectiveness of ultrasonicsampling was further tested using quantitative real-time PCR based onamplifying the 16S rDNA bacterial gene (FIG. 17B). Consistently,ultrasound collected 1.7×10⁴ bacteria/cm² which is significantly higherthan swabbing (4.5×10³ bacteria/cm²), and equivalent to scrubbingtechnique (1.6×10⁴ bacteria/cm²)

Example 11

Use of Sensitivity Enhancers To Facilitate Detection of Human IgE In LPM

The ability of sensitivity enhancers to facilitate detection of a modelanalyte—human IgE antibody—which was dissolved in a model LPM—1% w/vNLS-Brij 30 in a PBS, was tested. ELISA assay was used to evaluatedetection of human IgE antibody in presence or absence of sensitivityenhancers in LPM. Specifically, 1 microgram of antibodies (A80-108A,Bethyl laboratory, TX) with specific binding to human IgE antibodies wascoated per well of a 96-well ELISA plate. Human IgE (RC80-108, Bethyllaboratory, TX) was dissolved in the LPM with or without sensitivityenhancer at a concentration of 0-100 ng/ml. As a positive control, humanIgE samples were prepared by dissolving in a standard diluent containing1% NO; BSA and 0.05% w/v Tween 20 (P7949, Sigma-Aldrich, St. Louis, Mo.)in 50 mM tris-HCl-buffered saline (T6664, Sigma-Aldrich, St. Louis, Mo.)which is commonly used in immunoassays. Two types of sensitivityenhancers were formulated: 10% BSA and 0.5% Tween 20 in PBS and 10% BSAand 0.5% Tween 20 in 50 mM Tris-buffered saline. Each of thesesensitivity enhancers was separately added to LPM containing IgE in aratio of 1:10. After 30 minutes incubation of ELISA plates with astandard blocking buffer, these samples were incubated in individualwells for 1 hour. After washing the wells, HRP-conjugated-secondaryantibodies at a concentration of 1 microgram/ml were incubated in eachwell for 1 hour. After washing, a HRP-based chemiluminescence signal(induced by substrates 54-61-00, KPL, MD), signifying detection abilityof IgE antibodies by ELISA, was measured for each test case using aspectrophotometer.

FIG. 18 plots the chemiluminescence signal intensity from various testcases as a function of analyte concentration. Results show that LPM byitself was not a suitable detection reagent for ELISA assay as comparedwith positive control. However, adding sensitivity enhancers to LPMincreased the analytical assay's detection ability. Additionally,tris-buffered saline was shown to elevate the signal intensity ascompared with phosphate-buffered saline, when they are used as solventsto prepare sensitivity enhancers. These results demonstrated that LPM byitself is not efficient in facilitating analyte detection by ELISA;however, addition of sensitivity enhancers can significantly enhancedetection ability of analytes by ELISA.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofthe present invention is embodied by the appended claims.

Example 12

Delivery of Inulin and Acyclovir Into Pig Skin

Drug delivery experiments were performed on pig skin in vitro. Pre-cutfrozen full-thickness porcine skin, harvested from the lateral abdominalregion of Yorkshire pigs, was obtained from Lampire BiologicalLaboratory, Inc., PA. The skin was stored at -80° C. freezer prior tothe experiment. The skin was thawed at room temperature, and the skinwith no visible imperfections such as scratches and abrasions were cutinto small pieces (2.5×2.5 cm). Skin pieces were mounted on to a Franzdiffusion cell (PenneGear, Inc., PA). Before each experiment, thereceiver compartment was filled with a LPM or phosphate buffer saline(PBS). A 1%-w/v mixture of NLS and Brij 30 in PBS was chosen as a modelformulation of a LPM. Prior to each experiment, the electricalconductivity of the skin was measured to ensure its integrity. The skinwas considered damaged if the initial conductivity was more than 2.2microA/cm². Ultrasound was applied using a sonicator (VCX 400, Sonicsand Materials) operating at a frequency of 20 kHz at an intensity of 2.4W/cm² for 5 minutes. After the LPM or PBS was removed, the donorcompartment was filled with 10 microCi/ml solution of Inulin(NET086L001MC, PerkinElmer Life and Analytical Sciences, Inc., MA) inPBS. Samples were taken from the receiver compartments 24 hours afterultrasound application. In a separate experiment, a rotating abrasivesurface (a circular brush with plastic bristles) was introduced in thedonor chamber such that it directly contacted the skin sample. 10microCi/ml solution of Acyclovir was placed on the skin for 24 hours.The skin was washed by a saline and dissolved in Solvable (PerkinElmer,MA). The concentrations of those samples were measured by ascintillation counter (Tri-Carb 2100 TR, Packard, CT). All experimentswere conducted at room temperature, 22° C. Neither ultrasound norabrasive device was applied on the controls. Error bars indicate thestandard deviation.

Five minutes of ultrasound irradiation in combination with the LPMincreased drug transport, compared to that both by ultrasound alone andby the passive diffusion on intact skin, as shown in FIG. 19A. The sameeffect was observed when the skin was abraded with a moving brushingdevice comprising a plurality of bristles (FIG. 19B). In summary, theexamples using pig skin in vitro demonstrated that applying energy witha LPM is effective in enhancing the passage of molecules through or intotissues. Parameters such as power, time of application and a formulationof a LPM can be optimized to suit the individual situation, both withrespect to the type of tissue and the substances to be transported.

Although the present invention has been described in connection with thepreferred embodiments, it is to be understood that modifications andvariations may be utilized without departing from the principles andscope of the invention, as those skilled in the art will readilyunderstand. Accordingly, such modifications may be practiced within thescope of the following claims.

1. A kit for at least partly liquefying tissue, comprising: aliquefaction promoting medium (LPM), comprising: a non-ionic surfactant;a zwitterionic surfactant; and an abrasive material; instructions, theinstructions comprising directing a user to treat a tissue of a livingsubject by: applying the LPM together with the abrasive material to thetissue of the living subject; and transmitting energy to the tissue ofthe living subject through the abrasive material in the presence of theLPM effective to cause at least partial dissolution of one or morecomponents of the tissue of the living subject.
 2. The kit of claim 1,the instructions directing the user to treat the tissue of the livingsubject comprising one or more of: keratinized tissue, skin, and mucosalmembrane.
 3. The kit of claim 1, the instructions directing the user totreat the living subject or the tissue of the living subject that is oneof: healthy; or under the influence of one or more of: an allergy,urticaria, an auto-immune disease, a cardiovascular disease, cancer,diabetes, Alzheimer's, environmental contamination, a therapeutic drug,a drug of abuse, an infection, inflammation, psoriasis, atopicdermatitis, a scar, and an injury.
 4. The kit of claim 1, theinstructions directing the user to treat the tissue of the livingsubject comprising skin that is one or more of: normal, healthy, intact,dry, flaky, injured, damaged, shaven, allergic, scratched, scarred, andabraded.
 5. The kit of claim 1, the LPM comprising the non-ionicsurfactant and the zwitterionic surfactant together in a bufferedsolution.
 6. The kit of claim 1, the LPM being characterized by a totalconcentration (w/v) of the non-ionic surfactant and the zwitterionicsurfactant in a range of one or more of: between about 0.01% to about20%, between about 0.01% to about 10%, and between about 0.5% to about10%.
 7. The kit of claim 1, the LPM being characterized by a ratio ofthe non-ionic surfactant and the zwitterionic surfactant of betweenabout 25:75 and about 75:25.
 8. The kit of claim 1, the non-ionicsurfactant comprising one or more of: polyethylene glycol dodecyl ether,polyoxyethylene 23-lauryl ether, polyoxyethylene 2-cetyl ether,polyoxyethylene 10-cetyl ether, polyoxyethylene 20-cetyl ether,polyoxyethylene 2-stearyl ether, polyoxyethylene 10-stearyl ether,polyoxyethylene 20-stearyl ether, polyoxyethylene 2-oleyl ether,polyoxyethylene 10-oleyl ether, polyoxyethylene 100-stearyl ether, andpolyoxyethylene 21-stearyl ether.
 9. The kit of claim 1, thezwitterionic surfactant comprising one or more of: 3-(decyl dimethylammonia) propane sulfonate, 3-(dodecyl dimethyl ammonia) propanesulfonate, myristyldimethyl ammonia propane sulfonate, hexadecyldimethylammonia propane sulfonate, cocamidopropyl betaine, oleyl betaine,cocamidopropyl hydroxysultaine, and3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate.
 10. The kit ofclaim 1, the instructions directing the user to transmit the energy tothe tissue of the living subject in the presence of the LPM via one ormore of: stirring, abrasion, pressure, ultrasound, scraping, and shearforce.
 11. The kit of claim 1, the abrasive material comprising orcharacterized by one or more of: a fabric, quartz, a metal, a polymer,silica, silicon carbide, dust, alumina, a heterogeneous abrasive,abrasive crystals, diamond dust, a polymeric sponge, a natural sponge,an abrasive disc, an abrasive sheet, an abrasive ring, a scraper, and abrush.
 12. The kit of claim 1, the LPM further comprising the abrasivematerial, the abrasive material comprising one or more of: quartz, ametal, a polymer, silica, silicon carbide, dust, alumina, aheterogeneous abrasive, abrasive crystals, and diamond dust.
 13. The kitof claim 12, the LPM comprising the abrasive material in a concentrationrange of 0.01-99% (w/v).
 14. The kit of claim 1, further comprising areservoir, the instructions further directing the user to apply the LPMfrom the reservoir to the tissue of the living subject.
 15. The kit ofclaim 1, further comprising a drug, the instructions further comprisingdirecting the user to treat the living subject by contacting the drug tothe tissue of the living subject.
 16. The kit of claim 15, the drugcomprising one or more of: a protein, a peptide, a nucleic acidmolecule, an anti-inflammatory, an antiviral, an antifungal, anantibiotic, a local anesthetic, an antibody, a free-radical scavenger,an antioxidant, and a saccharide.
 17. The kit of claim 16, the drugcomprising a bioactive agent for therapeutic treatment of a condition inthe subject, the condition comprising one or more of: an allergy,urticaria, an auto-immune disease, a cardiovascular disease, cancer,diabetes, Alzheimer's, environmental contamination, drug abuse, aninfection, inflammation, psoriasis, atopic dermatitis, a scar, and aninjury.
 18. A kit for at least partly liquefying tissue, comprising: aliquefaction promoting medium (LPM) comprising: an abrasive material ina concentration range in the LPM of 0.01-99% (w/v); and 3-(decyldimethyl ammonia) propane sulfonate and polyethylene glycol dodecylether in a total surfactant concentration in the LPM of 0.01-20% (w/v);instructions, the instructions comprising directing a user to treat atissue of a living subject by: applying the LPM to the tissue of theliving subject, the tissue comprising at least one of skin and mucosalmembrane; and transmitting mechanical energy through the abrasivematerial to the tissue of the living subject in the presence of the LPM,the kit being effective to cause at least partial dissolution of one ormore components of the tissue of the living subject.
 19. The kit ofclaim 18, the instructions directing the user to treat the tissue of theliving subject comprising skin that is one or more of: normal, healthy,intact, dry, flaky, injured, damaged, shaven, allergic, scratched,scarred, and abraded.
 20. The kit of claim 18, the instructionsdirecting the user to treat the living subject or the tissue of theliving subject that is one of: healthy; or under the influence of one ormore of: an allergy, urticaria, an auto-immune disease, a cardiovasculardisease, cancer, diabetes, Alzheimer's, environmental contamination, atherapeutic drug, a drug of abuse, an infection, inflammation,psoriasis, atopic dermatitis, a scar, and an injury.